{"id":10711,"date":"2026-04-09T15:00:10","date_gmt":"2026-04-09T15:00:10","guid":{"rendered":"https:\/\/www.proefschriftmaken.nl\/portfolio\/siyu-sun\/"},"modified":"2026-04-23T07:18:52","modified_gmt":"2026-04-23T07:18:52","slug":"siyu-sun","status":"publish","type":"us_portfolio","link":"https:\/\/www.proefschriftmaken.nl\/en\/portfolio\/siyu-sun\/","title":{"rendered":"Siyu Sun"},"content":{"rendered":"","protected":false},"excerpt":{"rendered":"","protected":false},"author":8,"featured_media":12500,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"us_portfolio_category":[45],"class_list":["post-10711","us_portfolio","type-us_portfolio","status-publish","has-post-thumbnail","hentry","us_portfolio_category-new-template"],"acf":{"naam_van_het_proefschift":"Platelets, Red Blood Cells, Fibrinogen and Endothelial Cells: Essential Components in Blood Clotting","samenvatting":"In dit proefschrift heb ik mij geconcentreerd op een aantal complexe stollingsprocessen, om daarbij de rol van plaatjes, rode bloedcellen, fibrine en endotheelcellen te ontrafelen in de context van hemostase en trombose. Hoofdstuk 1 biedt een algemene inleiding, waarin de belangrijkste routes van het stollingsproces en hun relevantie worden gepresenteerd. Verder worden plaatjesreceptoren en signaalmechanismen ge\u00efntroduceerd, in het bijzonder de collageen- en fibrine(ogeen)-receptor glycoprote\u00efne VI (GPVI) samen met integrine \u03b1IIb\u03b23. Ook wordt achtergrondkennis gegeven over de invloed van andere bloedcellen in relatie tot bloedstolling, (beperkingen van) de meting van trombinegeneratie, als een integratieve stollingstest, en informatie over nieuwe van kameelachtigen afgeleide nanobodies met een enkel domein voor het bestuderen van de plaatjes- en coagulatieactivering.\n\nHoofdstuk 2 geeft een overzicht van bekende moleculaire oorzaken en gevolgen van immuunge\u00efnduceerde trombocytopenie (dat wil zeggen een laag plaatjesaantal in bloed) als meest voorkomende vormen van auto-immuunziekten. De beschreven aandoeningen omvatten idiopathische en trombotische trombocytopenische purpura, systemische lupus erythematosus, antifosfolipidensyndroom, drug-ge\u00efnduceerde trombocytopenie, heparine-afhankelijke trombocytopenie, door Covid-19-vaccin ge\u00efnduceerde trombose met trombocytopenie en het zogenaamde HELLP-syndroom (hemolyse, verhoogde leverenzymen en lage plaatjes). Voor elke ziekte of syndroom beschrijven we de plaatjesreceptoren voor de betreffende auto-antilichamen, de betrokken immuuncomplexen en evidentie voor de pathogene effecten van schadege\u00efnduceerde moleculaire patronen en complementfactoren. Daarnaast geven we aan hoe circulerende plaatjes kunnen dienen als een reservoir van immunomodulerende moleculen. We concluderen dat er mogelijk een centrale rol is weggelegd voor de Fc\u03b3RIIA-receptoren op plaatjes, maar in welke mate is nog onduidelijk. De door antilichamen geactiveerde plaatjes moduleren op hun beurt het aangeboren en adaptieve immuunsysteem door meerdere pro-inflammatoire en immuunmediatoren vrij te zetten. Duidelijk is dat de besproken plaatjes-afhankelijke processen kunnen leiden tot trombocytopenie, maar ook tot tromboses en bloedingen.\n\nIn Hoofdstuk 3 beoogden we de signaalroutes op te helderen die differenti\u00ebren tussen een permanente of transi\u00ebnte activeringstoestand van plaatjes. We onderzochten de routes opgewekt door GPVI en door de protease-geactiveerde receptoren (PAR1\/4) voor trombine, steeds op het vermogen om een tijdsafhankelijke integrine \u03b1IIb\u03b23-activering en plaatjesaggregatie te induceren. We vonden dat voorbehandeling van de cellen met farmacologische remmers de integrine \u03b1IIb\u03b23-activering onderdrukte (samen met P-selectine-expressie) in de target-volgorde van prote\u00efnekinase C (PKC) > glycogeensynthasekinase 3 > \u03b2-arrestine > fosfatidylinositol-3-kinase. Nabehandeling met remmers leidde tot secundaire inactivering van \u03b1IIb\u03b23 (niet van P-selectine-expressie) in dezelfde volgorde, maar hier bleef de omkeerbaarheid beperkt tot GPVI- en PAR1-agonisten. Voor- en nabehandeling van plaatjes met ticagrelor, een blokker van de P2Y12-receptoren voor ADP, veroorzaakte inactivering van het integrine. Plaatjesspreidingstesten toonden aan dat de blokkering van PKC of P2Y12 gedeeltelijk leidde tot de overgang van een meer geactiveerde naar een meer rustende plaatjesmorfologie. We concludeerden dat zowel PKC- als autocriene ADP-signalering bijdragen aan een persistente \u03b1IIb\u03b23-activering en daarmee aan een stabiele plaatjesaggregatie. Deze bevindingen zijn relevant voor de optimalisatie van de behandeling met aggregatieremmers.\n\nHoofdstuk 4 betreft een onderzoek naar het stollingsvermogen van rode bloedcellen en plaatjes in de context van bloedingen en trombotische aandoeningen. Met behulp van een nieuwe high-throughput-methode voor trombinegeneratie in volbloed, zulks ter vergelijking met autoloog plaatjesrijk plasma, konden we de bijdragen van alle bloedcellen aan het stollingsproces vastleggen. We noteerden een snellere en aanvankelijk hogere generatie van trombine en een kortere stollingstijd in volbloed dan in plaatjesrijk plasma. Dit was het geval voor een reeks triggers, waaronder weefselfactor, Russell's addergif, factor Xa, factor XIa en trombine. De trombinegeneratie versnelde met het hematocrietniveau, maar werd vertraagd na voorbehandeling van de rode bloedcellen met fosfatidylserine-blokkerend annexine A5. In reconstitutie bloedmonsters verhoogde de blokkering van fosfatidylserine op rode bloedcellen de GPVI-afhankelijke plaatjesprocoagulante activiteit. Bij pati\u00ebnten met bloedarmoede of erytrocytose vonden we middels clusteranalyse verschillende trombinegeneratieprofielen, die echter nog steeds afhankelijk waren van het fosfatidylserine op rode bloedcellen. We concludeerden dat zowel rode bloedcellen als plaatjes, op een fosfatidylserine-afhankelijke manier, bijdragen tot de generatie van trombine. Bepaling van de hypo- of hyperstollingsactiviteit in volbloed kan helpen bij het karakteriseren van een bloedings- of tromboserisico.\n\nIn Hoofdstuk 5 richtten we ons op de bijdrage van fibrinogeen en fibrine in plasma aan het stollingsproces. We beschrijven de effecten van een nieuwe nanobody, Nb106, dat gericht is tegen fibrine, maar niet tegen fibrinogeen. In volbloed of plasma verlaagde Nb106 dosisafhankelijk de trombinegeneratie tot 50%, evenwel zonder de kinetiek te be\u00efnvloeden. Relevant was dat Nb106 niet langer effectief was in de afwezigheid van fibrinogeen of antitrombine, maar dat het de trombinegeneratie bleef blokkeren in de afwezigheid van factoren IX-XII, trombomoduline, geactiveerd prote\u00efne C of orale antistollingsmiddelen. We concludeerden dat er een aanzienlijke pool van proteolytisch actief, aan fibrine gebonden trombine gevormd wordt, die beschermt tegen antitrombine-inactivering. Verdere experimenten gaven aan dat het fibrine-gebonden trombine verantwoordelijk is voor een correcte vorming van fibrinevezels. Bij afwezigheid hiervan, met Nb106, vervormen de vezels tot een samengeklonterde structuur. Bovendien is deze pool nodig voor de clot-retractie en de fibrinevorming onder stromingscondities bij lage afschuifsnelheid. De trombinegeneratie werd minder onderdrukt door Nb106 in plasma's van pati\u00ebnten met congenitale dysfibrinogenemie, wanneer vergeleken met de plasma's van een cohort van controlepersonen. Gezamenlijk benadrukken deze bevindingen de functie van fibrine-gebonden trombine voor een goede stolselvorming en maken dit trombine tot een potentieel therapeutisch doelwit bij bloed-gerelateerde ziekten.\n\nIn Hoofdstuk 6 konden we aantonen dat een verhoging van het fibrinogeenniveau de GPVI-ge\u00efnduceerde activatie van plaatjes verhoogt in termen van P-selectine-expressie, vooral bij lage agonistdoses. In overeenstemming met deze bevinding bleek dat fibrinogeen ook dosisafhankelijk het proces van trombinegeneratie in plaatjesrijk plasma verhoogt, zowel in de aanwezigheid als afwezigheid van een externe GPVI-agonist. Deze fibrinogeeneffecten waren afwezig met de anti-GPVI-nanobody Nb2. Eerder was aangetoond dat bij pati\u00ebnten met een prote\u00efne C-defici\u00ebntie \u2013 in sterkere mate dan bij een prote\u00efne S-defici\u00ebntie \u2013 de GPVI-afhankelijke trombus- en fibrinevorming verminderd is, in vergelijking met gezonde proefpersonen. We wilden nu vaststellen of fibrinogeen in het pati\u00ebntenplasma de GPVI-activeringsmodulator is. In deze plasma's aangevuld met donorplaatjes konden we inderdaad een verminderd GPVI-afhankelijk effect meten op de trombinegeneratie bij prote\u00efne C-defici\u00ebntie, maar niet bij prote\u00efne S-defici\u00ebntie. Deze verlaging bleek echter onafhankelijk te zijn van het fibrinogeenniveau.\n\nHoofdstuk 7 betreft een onderzoek naar de effecten van endotheelcellen op het trombinegeneratieproces, zowel in volbloed als in plaatjesrijk plasma. Opvallend was dat de aanwezigheid van een endotheliale monolaag de generatie van trombine in beide situaties vertraagde en nagenoeg volledig onderdrukte. Mechanistisch vonden we dat de blokkering van de heparine-achtige proteoglycanen met polybreen of de afwezigheid van het heparine-afhankelijke antitrombine de endotheliale antistollingsactiviteit tenietdeed. Bovendien leidde behandeling van de cellen met andexanet-\u03b1 of met antilichamen tegen TFPI (tissue factor pathway inhibitor) tot een vermindering van het endotheel-effect. In aanmerking nemend de deels additieve rol van antitrombine en TFPI bij de stollingsregulering, wijzen onze gegevens dus op een functionele niet-redundantie van de endotheliale antistollingsactiviteit, op een wijze waarin zowel proteoglycanen als TFPI bijdragen tot de trombineremming.\n\nIn Hoofdstuk 8 bespreek ik de belangrijkste bevindingen en bijdragen van dit proefschrift. Mijn conclusie is dat de huidige onderzoeken naar plaatjes, rode bloedcellen, fibrine en endotheelcellen aanvullende inzichten en routes bieden binnen het concept van celgebaseerde hemostase en trombose.\n\n\u4e2d\u6587\u6982\u8ff0\n\n\u5728\u672c\u8bba\u6587\u4e2d\uff0c\u6211\u4eec\u4e3b\u8981\u7814\u7a76\u4e86\u590d\u6742\u7684\u51dd\u8840\u8fc7\u7a0b\uff0c\u5305\u62ec\u8840\u6d46\u51dd\u8840\u56e0\u5b50\u3001\u8840\u5c0f\u677f\u3001\u7ea2\u7ec6\u80de\u3001\u7ea4\u7ef4\u86cb\u767d\u548c\u5185\u76ae\u7ec6\u80de\u5bf9\u51dd\u8840\u8fc7\u7a0b\u7684\u5f71\u54cd\u3002\u7b2c 1 \u7ae0\u6982\u8ff0\u4e86\u4e0d\u65ad\u53d8\u5316\u7684\u5371\u9669\u56e0\u7d20\u5bf9\u51dd\u8840\u7684\u5f71\u54cd\uff0c\u5305\u62ec\u8840\u5c0f\u677f\u53d7\u4f53\u548c\u6b62\u8840\u548c\u8840\u6813\u5f62\u6210\u4e2d\u7684\u4fe1\u53f7\u4f20\u5bfc\u3001\u51dd\u8840\u6982\u8ff0\u3001\u8840\u7ec6\u80de\u5bf9\u51dd\u8840\u7684\u5f71\u54cd\u3001\u51dd\u8840\u9176\u751f\u6210\u6d4b\u5b9a\u548c\u9a86\u9a7c\u6e90\u5355\u57df\u6297\u4f53\u5728\u6b62\u8840\u4e2d\u7684\u4f5c\u7528\u3002\n\n\u7b2c 2 \u7ae0\u6982\u8ff0\u4e86\u6700\u5e38\u89c1\u7684\u81ea\u8eab\u514d\u75ab\u6027\u75be\u75c5\u4e2d IIT \u7684\u5206\u5b50\u673a\u5236\u548c\u540e\u679c\u3002\u8fd9\u4e9b\u75be\u75c5\u5305\u62ec\u7279\u53d1\u6027\u8840\u5c0f\u677f\u51cf\u5c11\u6027\u7d2b\u765c\uff08ITP\uff09\u3001\u7cfb\u7edf\u6027\u7ea2\u6591\u72fc\u75ae\uff08SLE\uff09\u3001\u6297\u78f7\u8102\u7efc\u5408\u5f81\uff08APS\uff09\u3001\u836f\u7269\u8bf1\u5bfc\u7684\u8840\u5c0f\u677f\u51cf\u5c11\u75c7\uff08DITP\uff09\u3001\u809d\u7d20\u8bf1\u5bfc\u7684\u8840\u5c0f\u677f\u51cf\u5c11\u75c7\uff08HIT\uff09\u3001Covid-19 \u75ab\u82d7\u8bf1\u5bfc\u7684\u8840\u6813\u5f62\u6210\u4f34\u8840\u5c0f\u677f\u51cf\u5c11\u75c7\uff08VITT\uff09\u3001\u8840\u6813\u6027\u8840\u5c0f\u677f\u51cf\u5c11\u6027\u7d2b\u765c\uff08TTP\uff09\u4ee5\u53ca\u6eb6\u8840\u3001\u809d\u9176\u5347\u9ad8\u548c\u8840\u5c0f\u677f\u8fc7\u4f4e\uff08HELLP\uff09\u7efc\u5408\u5f81\u3002\u6211\u4eec\u7684\u91cd\u70b9\u662f\u4e0e\u81ea\u8eab\u6297\u4f53\u3001\u514d\u75ab\u590d\u5408\u7269\u3001\u635f\u4f24\u76f8\u5173\u5206\u5b50\u6a21\u5f0f\uff08DAMPs\uff09\u548c\u8865\u4f53\u56e0\u5b50\u7ed3\u5408\u7684\u8840\u5c0f\u677f\u53d7\u4f53\u3002\u6b64\u5916\uff0c\u6211\u4eec\u8fd8\u56de\u987e\u4e86\u8840\u6db2\u5faa\u73af\u4e2d\u7684\u8840\u5c0f\u677f\u662f\u5982\u4f55\u5145\u5f53\u514d\u75ab\u8c03\u8282\u5206\u5b50\u5e93\u3002\u901a\u8fc7\u66f4\u65b0\u8840\u5c0f\u677f\u5728\u81ea\u8eab\u514d\u75ab\u6027\u75be\u75c5\u53d1\u75c5\u673a\u5236\u4e2d\u7684\u5206\u5b50\u673a\u5236\u548c\u4f5c\u7528\uff0c\u6211\u4eec\u5f3a\u8c03\u4e86\u57fa\u4e8e\u8840\u5c0f\u677f\u7684\u9014\u5f84\u53ef\u80fd\u5bfc\u81f4\u8840\u5c0f\u677f\u51cf\u5c11\uff0c\u5e76\u4e0e\u8840\u6813\u6216\u51fa\u8840\u4e8b\u4ef6\u6709\u5173\u3002\n\n\u4e3a\u4e86\u9610\u660e\u533a\u5206\u8840\u5c0f\u677f\u6c38\u4e45\u6216\u77ed\u6682\u6d3b\u5316\u72b6\u6001\u7684\u4fe1\u53f7\u901a\u8def\u3002\u5728\u7b2c\u4e09\u7ae0\u4e2d\uff0c\u6211\u4eec\u63a2\u8ba8\u4e86\u7531\u80f6\u539f\u53d7\u4f53\u7cd6\u86cb\u767d VI\uff08GPVI\uff09\u6216\u51dd\u8840\u9176\u86cb\u767d\u9176\u6d3b\u5316\u53d7\u4f53\uff08PAR\uff09\u8bf1\u5bfc\u7684\u8840\u5c0f\u677f\u4fe1\u53f7\u4f20\u5bfc\u673a\u5236\uff0c\u8fd9\u4e9b\u673a\u5236\u53ef\u8c03\u8282\u65f6\u95f4\u4f9d\u8d56\u6027 \u03b1IIb\u03b23 \u6d3b\u5316\u3002\u6211\u4eec\u53d1\u73b0\uff0c\u7528\u836f\u7269\u6291\u5236\u5242\u9884\u5904\u7406\u8840\u5c0f\u677f\u53ef\u51cf\u5c11 GPVI \u548c PAR \u8bf1\u5bfc\u7684\u6574\u5408\u7d20 \u03b1IIb\u03b23 \u6fc0\u6d3b\u548c P \u9009\u62e9\u7d20\u8868\u8fbe\uff0c\u5176\u987a\u5e8f\u4e3a\u86cb\u767d\u6fc0\u9176 C (PKC) > \u7cd6\u539f\u5408\u9176\u6fc0\u9176 3 > \u03b2-\u963f\u53f8\u5339\u6797 > \u78f7\u8102\u9170\u808c\u9187-3-\u6fc0\u9176\u3002\u540e\u5904\u7406\u663e\u793a\u7ee7\u53d1\u6027 \u03b1IIb\u03b23 \u5931\u6d3b\uff08\u800c\u975e P-\u9009\u62e9\u7d20\u8868\u8fbe\uff09\uff0c\u987a\u5e8f\u76f8\u540c\uff0c\u4f46\u8fd9\u79cd\u53ef\u9006\u6027\u4ec5\u9650\u4e8e CRP \u548c PAR1 \u6fc0\u52a8\u5242\u3002\u8054\u5408\u6291\u5236\u4f20\u7edf\u548c\u65b0\u578b PKC \u540c\u5de5\u9176\u5bf9\u6574\u5408\u7d20\u5c01\u95ed\u6700\u6709\u6548\u3002\u963b\u65ad P2Y12 ADP \u53d7\u4f53\u7684\u66ff\u5361\u683c\u96f7\uff08ticagrelor\uff09\u53ef\u589e\u5f3a \u03b1IIb\u03b23 \u7684\u5931\u6d3b\u3002\u94fa\u5c55\u8bd5\u9a8c\u8868\u660e\uff0cPKC \u6216 P2Y12 \u6291\u5236\u53ef\u4f7f\u8840\u5c0f\u677f\u4ece\u7247\u72b6\u90e8\u5206\u8f6c\u53d8\u4e3a\u66f4\u5706\u76d8\u72b6\u3002\u6211\u4eec\u7684\u7ed3\u8bba\u662f\uff0cPKC \u548c\u81ea\u5206\u6ccc ADP \u4fe1\u53f7\u6709\u52a9\u4e8e\u6574\u5408\u7d20 \u03b1\u2161b\u03b23 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\u5728\u51dd\u8840\u63a7\u5236\u4e2d\u7684\u4e0d\u540c\u4f5c\u7528\uff0c\u6211\u4eec\u7684\u6570\u636e\u8868\u660e\u5185\u76ae\u6297\u51dd\u6d3b\u6027\u5b58\u5728\u529f\u80fd\u975e\u5197\u4f59\u6027\uff0c\u5373\u86cb\u767d\u805a\u7cd6\u548c\u8868\u9762\u8868\u8fbe\u7684 TFPI \u90fd\u6709\u52a9\u4e8e\u51dd\u8840\u9176\u6291\u5236\u7684\u4f5c\u7528\u3002\n\n\u5728\u7b2c 8 \u7ae0\u4e2d\uff0c\u6211\u4eec\u8ba8\u8bba\u4e86\u672c\u8bba\u6587\u7684\u4e3b\u8981\u53d1\u73b0\u548c\u8d21\u732e\u3002\u53ef\u4ee5\u9884\u89c1\u7684\u662f\uff0c\u76ee\u524d\u5bf9\u8840\u5c0f\u677f\u3001\u7ea2\u7ec6\u80de\u3001\u7ea4\u7ef4\u86cb\u767d\u548c\u5185\u76ae\u7ec6\u80de\u7684\u7814\u7a76\u4e3a\u6b62\u8840\u548c\u8840\u6813\u5f62\u6210\u63d0\u4f9b\u4e86\u66f4\u591a\u7684\u89c1\u89e3\u3002\n\nIMPACT\n\nCardiovascular diseases (CVD) are a leading cause of death globally, taking away an estimated 15 million lives each year. Patients with CVD are in the need of a proper diagnosis and an effective therapy to manage and return the progress of their disease and suffering. Activation of platelets and the coagulation system are the key targets of treatment in CVD. My research has focused on unravelling the intricate interplay between coagulation factors, platelets, red blood cells, endothelial cells and fibrinogen in the process of blood coagulation, striving to new insights with the potential to improve fundamental or clinical practice in the field of haemostasis and thrombosis.\n\nTarget group\nThe primary target groups include healthcare professionals, particularly clinicians involved in diagnosing and treating haemostatic disorders and cardiovascular diseases. Additionally, our findings will have broad relevance to biomedical researchers who focus on understanding the intricacies of blood coagulation, including those in academic institutions, in research laboratories, and in pharmaceutical as well as blood disease diagnostic companies. Those individuals are involved in developing novel therapies and coagulation testing for haemostatic disorders and cardiovascular diseases.\n\nInnovation and implementation\nBy elucidating the dynamic interactions among coagulation factors, platelets, red blood cells, endothelial cells and fibrin during the coagulation process, it will be possible to further develop diagnostic tools and therapeutic strategies. These include the optimisation of advanced whole blood thrombin generation assay to evaluate thrombin dynamics in vitro, as well as seeking for novel targeted therapies that modulate platelets responses and the fibrin part of the coagulation cascade.\n\nConsidering the immunity role of platelets, I explored how platelets are activated and destructed in several immune-induced thrombocytopenia diseases. It is furthermore shown that platelet stimulation via glycoprotein VI (GPVI) or thrombin receptor PAR1 causes transient integrin \u03b1IIb\u03b23 activation, while stimulation via the PAR4 receptors leads to a more permanent \u03b1IIb\u03b23 activation. It appeared that the protein kinase C pathway is most crucial for integrin \u03b1IIb\u03b23 activation, and that autocrine responses via ADP receptors are essential for the sustained integrin \u03b1IIb\u03b23 opening. These observations will help to drive the development of antiplatelet medications.\n\nAnother relevant finding concerns the platelet GPVI co-activation via fibrinogen, an effect that is enhanced with the fibrinogen concentration. The results of this study can contribute to the development of novel and safer antithrombotic inhibitors that specifically target the GPVI-fibrinogen interaction.\n\nOur research findings also involve collaboration with clinicians to aim to apply the new method of whole blood thrombin generation for examining cohorts of patients with anaemia. Application of this method can ensure a timely assessment of the risk of bleeding or thrombosis, and result in personalised treatment approaches for patients with anaemia. Considering the clotting role of fibrin this, we used a novel anti-fibrin nanobody to explore how fibrin modulates thrombin generation. These findings in this context highlight a unique role of fibrin-bound thrombin, potentially serving as a therapeutic target in blood-borne diseases, while preserving haemostatic function. Furthermore, we have explored the potent anticoagulant role of endothelial cells in thrombin generation.\n\nSocial and economic relevance\nThis relevance of my research lies in the promise that advanced thrombin generation assays, including whole blood thrombin generation and endothelial-modulated thrombin generation, can improve the patient outcome and quality of life by a better diagnosis and disease management, such as thrombosis and haemorrhage, which pose significant morbidity and mortality risks globally. Furthermore, my research contributes to the advancement of precision medicine, paving the way for more effective and personalised interventions in coagulation-related disorders. By providing clinicians with a deeper understanding of how platelets, red blood cells, endothelial cells and fibrin drive coagulation, our research empowers them to make more informed treatment decisions. Ultimately, the development of targeted therapies based on an anti-GPVI nanobody and anti-fibrin nanobody has the potential to spur economic growth through the creation of new medical technologies and pharmaceutical products.\n\nPUBLICATIONS\n\n1. Sun S, Du X, Fu M, Khan AR, Ji J, Liu W, Zhai G. Galactosamine-modified PEG-PLA\/TPGS micelles for the oral delivery of curcumin. Int J Pharmaceutics, 2021, 595: 120227.\n2. Sun S, Zhang H, Wang X, He S, Zhai G. Development and evaluation of ibuprofen loaded mixed micelles preparations for topical delivery. J Drug Delivery Science Techn, 2018, 48: 363-371.\n3. Liu M, Zhang Y, Sun S, Khan AR, Ji J, Yang M, Zhai G. Recent advances in electrospun for drug delivery purpose. J Drug Targeting, 2019, 27(3): 270-282.\n4. Sun S, Urbanus RT, ten Cate H, de Groot PG, de Laat B, Heemskerk JWM, Roest M. Platelet activation mechanisms and consequences of immune thrombocytopenia. Cells. 2021;10(12):3386.\n5. Sun S, Campello E, Zou J, Konings J, Huskens D, Wan J, Fernandez D, Reutelingsperger C, ten Cate H, Toffanin S, Bulato C, de Groot PG, Simioni P, Heemskerk JWM, Roest M. Crucial roles of red blood cells and platelets in whole blood thrombin generation. Blood Adv. 2023;7(21):6717-6731.\n6. Zou J, Sun S, Simone I, ten Cate H, de Groot PG, de Laat B, Roest M, Heemskerk JWM, Swieringa F. Platelet activation pathways controlling reversible integrin \u03b1IIb\u03b23 activation. TH Open. 2024 Jun 22;8(2):e232-e242.\n7. Sun S, Urbanus R, Konings J, Campello E, Oftering P, Beck S, Bulato C, Kremers R, Huskens D, Swieringa F, Xiang G, Zou J, Nieswandt B, Simioni P, de Groot PG, Roest M, Heemskerk JWM. de Laat B. Thrombin pools in fibrin-independent and fibrin-dependent coagulation regulating clot structure. Preparation for submission.\n8. Sun S, Campello E, Bulato C, Tullemans B, Spiezia L, de Groot PG, Simioni P, Heemskerk JWM, de Laat B, Roest M. Fibrin(ogen) modulation and GPVI dependent platelet responses. To be submitted.\n9. Sch\u00f6nichen C, Sun S, Middelveld H, Huskens D, de Groot PG, Heemskerk JWM, Roest M, de Laat B. Functional non-redundancy of endothelial anti-thrombin generation mechanisms. Thromb Res. 2024 Dec:244:109208.\n10. Sch\u00f6nichen C, Silvia Nedwed A, Provenzale I, A. Solari F, Marini F, Sun S, Stoll M, de Laat B, Sickmann A, J.E. Kuijpers M, Heemskerk JWM, Jurk K. Impaired antiplatelet and anticoagulant functions of endothelial cells by proinflammatory stimuli: a multi-omics approach. To be submitted.\n11. M.S. Hoornstra I, Zhao J, Heemskerk JWM, Sun S, Sch\u00f6nichen C, Middelveld H, Heylen R, de Laat B, Roest M. Andexanet alpha is a potent DOAC reversal agent with pro-coagulant adverse effects. Preparation for submission.\n\nPRESENTATIONS AT CONFERENCES\n\n1. Sun S, Zou J, Konings J, Huskens D, Wan J, Reutelingsperger CPM, ten Cate H, de Groot PG, de Laat B, Heemskerk JWM, Roest M. Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation. 2022 July, International Society on Thrombosis and Haemostasis (ISTH) annual congress, London, UK. Poster presentation.\n2. Zou J, Sun S, De Simone I, ten Cate H, de Groot PG, Heemskerk JWM, de Laat B, Roest M. Establishing the signalling pathways regulating reversible platelet integrin activation. 2022 July, International Society on Thrombosis and Haemostasis (ISTH) annual congress, London, UK. Poster presentation.\n3. Sun S. Campello E, Zou J, Konings J, Huskens D, Wan J, Fernandez D, Reutelingsperger CPM, ten Cate H, Toffanin S, Bulato C, de Groot PG, de Laat B, Simioni P, Heemskerk JWM, Roest M. Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation. 2023 June, ISTH annual congress, Montr\u00e9al, Canada. Oral presentation.\n4. Sun S, Zou J, Konings J, Huskens D, Wan J, Reutelingsperger CPM, ten Cate H, de Groot PG, de Laat B, Heemskerk JWM, Roest M. Complementary roles of red blood cells and platelets in high-throughput whole-blood thrombin generation. 2023 September, 6th EUPLAN International Conference, Bristol, UK. Poster presentation.\n5. Sun S, Konings J, Urbanus RT, Huskens D, Swieringa F, de Laat-Kremers R, Zou J, de Groot PG, Roest M, Heemskerk JWM, de Laat B. Diversity of plasmatic thrombin pools regulate thrombin generation and blood clotting: interference by a novel nanobody. 2023 December, 65th ASH Annual Meeting & Exposition, San Diego, CA, USA. Poster presentation.\n6. Sun S, Konings J, Urbanus RT, Huskens D, Swieringa F, de Laat-Kremers R, Zou J, de Groot PG, Roest M, Heemskerk JWM, de Laat B. Concurrent interference of thrombin and fibrin fiber generation by an anti-fibrin nanobody in human and mouse plasma. 2024 February, 68th GTH Annual Meeting, Vienna, Austria. Poster presentation.\n7. Sun S, Konings J, Urbanus RT, Huskens D, Swieringa F, de Laat-Kremers R, Zou J, de Groot PG, Roest M, Heemskerk JWM, de Laat B. Diversity of plasmatic thrombin pools regulate thrombin generation and blood clotting: interference by a novel nanobody. 2024 February, 68th GTH Annual Meeting, Vienna, Austria. Oral presentation.\n8. Sun S, Feller T, D. Connell S, Konings J, Urbanus RT, Huskens D, de Groot PG, Roest M, Heemskerk JWM, A. S. Ari\u00ebns R, de Laat B. Fibrin clot structure and function dependent on thrombin modulation with a novel anti-fibrin nanobody. 2025 January, BSHT-NVTH Joint ASM, Newcastle upon Tyne, UK. Oral presentation. Excellence Award.","summary":"In this thesis, I focus on several of the complex and intricate coagulation processes, to better know the roles of platelets, red blood cells, fibrin and endothelial cells in the context of haemostasis and thrombosis. Chapter 1 provides a general introduction presenting the main pathways of the coagulation process and their importance. Platelet receptors and signalling mechanisms were furthermore introduced, in particular the collagen and fibrin(ogen) receptor glycoprotein VI (GPVI) together with integrin \u03b1IIb\u03b23. Also given is background knowledge on the influence of other blood cells in relation to coagulation, (limitations of) the measurements of thrombin generation, as an integrative coagulation assay, and information on novel camelid-derived single-domain nanobodies for studying platelet and coagulation activation.\n\nChapter 2 presents an overview of the reported molecular causes and consequences of immune-induced thrombocytopenia (i.e. low platelet counts) as most common forms of autoimmune disorders. The disorders described include idiopathic and thrombotic thrombocytopenic purpura, systemic lupus erythematosus, antiphospholipid syndrome, drug-induced thrombocytopenia, heparin-induced thrombocytopenia, Covid-19 vaccine-induced thrombosis with thrombocytopenia, and the so-called HELLP syndrome (haemolysis, elevated liver enzymes and low platelets). For each disease or syndrome, we review the platelet receptors that bind the inducing auto-antibodies, the involved immune complexes, and the evidence for pathogenic effects of damage associated molecular patterns and complement factors. In addition, we review how circulating platelets can serve as a reservoir of immunomodulatory molecules. We conclude that there possibly is a central role of the platelet Fc\u03b3RIIA receptors, but to which extent is still unclear. The antibody-activated platelets in turn modulate the innate and adaptive immune systems by releasing multiple pro-inflammatory and immune mediators. We highlight that the discussed platelet-based pathways that can predispose for thrombocytopenia as well as thrombotic and bleeding events.\n\nIn Chapter 3 we aim to elucidate the signalling pathways that differentiate between a permanent or transient activation state of platelets. We explored the pathways induced by GPVI and by the protease-activated receptors (PAR1\/4) for thrombin, in each case for their ability to induce time-dependent integrin \u03b1IIb\u03b23 activation and platelet aggregation. We found that pretreatment of the cells with given pharmacological inhibitors suppressed the agonist-induced \u03b1IIb\u03b23 activation (along with P-selectin expression) in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > \u03b2-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed a secondary \u03b1IIb\u03b23 inactivation (not P-selectin expression) in the same order, but the reversibility was confined to GPVI and PAR1 agonists. Platelet pre- and posttreatment with ticagrelor, blocking the P2Y12 receptors for ADP, caused inactivation of the integrin. Platelet spreading assays showed that the blockage of either PKC or P2Y12 partially induced the conversion from a more activated to a more resting platelet shape. We concluded that both PKC and autocrine ADP signalling contribute to a persistent integrin \u03b1IIb\u03b23 activation and hence a stable platelet aggregation. These findings are relevant for the optimisation of antiplatelet treatment.\n\nChapter 4 concentrates on the coagulation capacity of red blood cells and platelets in the context of bleeding and thrombotic disorders. Using a new high-throughput method of whole-blood thrombin generation, such in comparison to autologous platelet-rich plasma, we could define the contributions of all blood cell to the clotting process. We observed a faster and initially higher generation of thrombin and a shorter coagulation time in whole blood than in platelet-rich plasma. This was the case for a range of low coagulant triggers, including tissue factor, Russell's viper venom, factor Xa, factor XIa and thrombin. Thrombin generation accelerated with the hematocrit level but delayed after prior treatment of red blood cells with the phosphatidylserine-blocking annexin A5. In reconstituted blood samples, a prior selective blockage of phosphatidylserine on red blood cells enhanced the GPVI-dependent platelet procoagulant activity. In patients with anaemia or erythrocytosis, cluster analysis revealed distinct thrombin generation profiles, which still were dependent on phosphatidylserine exposure of the red cells. We concluded that both red blood cells and platelets, in a phosphatidylserine-dependent way, contribute to the thrombin generation. Determination of the whole blood hypo-or hyper-coagulant activity may help to characterise a bleeding or thrombosis risk.\n\nThe topic of Chapter 5 is assessment of the contribution of plasma fibrinogen and fibrin to the clotting process. We describe the effects of a novel nanobody, Nb106, which recognises an epitope on fibrin but not on fibrinogen. In whole blood or plasma, Nb106 dose-dependently lowered the tissue factor-induced thrombin generation up to 50%, without affecting the kinetics. Importantly, Nb106 was no longer effective in the absence of fibrinogen or antithrombin, while it continued to block thrombin generation in the absence of factors IX-XII, thrombomodulin, activated protein C or oral anticoagulants. We concluded that there exist a considerable pool of proteolytically active, fibrin-bound thrombin that is protected for antithrombin inactivation. Further experiments indicated that this fibrin-bound pool of thrombin is responsible for the formation of fibrin fibers. In the absence of this pool, with Nb106 present, the fibrin fibers distort to clumped structures. Moreover, it is needed for clot retraction and proper flow-dependent fibrin formation at low shear rate. Thrombin generation was less suppressed by Nb106 in plasmas from patients with congenital dysfibrinogenemia, when compared to plasmas from a cohort of control subjects. Collectively, these findings highlight the role of fibrin-bound thrombin in proper clot formation and make it a potential therapeutic target in blood-borne diseases.\n\nIn Chapter 6, we observed that an increase in fibrinogen level enhanced GPVI-induced platelet activation in terms of P-selectin expression, in particular at low agonist doses. In support of this finding, we determined that fibrinogen dose-dependently increased the process of thrombin generation in platelet-rich plasma both in the presence and absence of external GPVI agonist. These fibrinogen effects were antagonised by the anti-GPVI nanobody Nb2. Prior evidence was obtained that in patients with a protein C deficiency - to a larger extent than with a protein S deficiency - GPVI-dependent thrombus and fibrin formation is impaired, in comparison to healthy subjects. We now determined whether fibrinogen in the patients' plasma was the GPVI activation modulator. In the plasmas supplemented with donor platelets, we indeed measured a reduced GPVI-dependent effect on thrombin generation in cases of protein C deficiency, but not of protein S deficiency. However, this reduction appeared to be independent of the fibrinogen level.\n\nIn Chapter 7 it is investigated the effect of endothelial cells on the thrombin generation process either in whole blood and in platelet-containing plasma. Strikingly, the presence of an endothelial monolayer delayed and almost fully abrogated thrombin generation in both settings. Mechanistically, we found that the blocking of heparin-like proteoglycans with polybrene or absence of the heparin-effecting antithrombin reverted the endothelial anticoagulant activity. In addition, treatment of the cells with andexanet-\u03b1 or with antibodies against tissue factor pathway inhibitor (TFPI) also in part reverted the endothelial effect. Taken into consideration the partly additive roles of antithrombin and TFPI in coagulation regulation, our data hence point to a functional non-redundancy of the endothelial anticoagulant activity, in the way that both proteoglycans and surface-expressed TFPI contribute to the repertoire of thrombin inhibition.\n\nIn Chapter 8, I discuss the key findings and contributions of this thesis. My conclusion is that the current studies on platelets, red blood cells, fibrin and endothelial cells provide additional insights and pathways within the concept of cell-based haemostasis and thrombosis.","auteur":"Siyu Sun","auteur_slug":"siyu-sun","publicatiedatum":"24 maart 2025","taal":"EN","url_flipbook":"https:\/\/ebook.proefschriftmaken.nl\/ebook\/siyusun?iframe=true","url_download_pdf":"","url_epub":"","ordernummer":"FTP-202604091456","isbn":"978-94-6510-466-9","doi_nummer":"","naam_universiteit":"Universiteit Maastricht","afbeeldingen":12500,"naam_student:":"","binnenwerk":"","universiteit":"Universiteit Maastricht","cover":"","afwerking":"","cover_afwerking":"","design":""},"_links":{"self":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/10711","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio"}],"about":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/types\/us_portfolio"}],"author":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/comments?post=10711"}],"version-history":[{"count":1,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/10711\/revisions"}],"predecessor-version":[{"id":10714,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/10711\/revisions\/10714"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media\/12500"}],"wp:attachment":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media?parent=10711"}],"wp:term":[{"taxonomy":"us_portfolio_category","embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio_category?post=10711"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}