Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.     

Activated platelet-derived microparticles in thalassaemia

Thromboembolic complications have been documented in thalassaemia patients. The aggregability of abnormal red blood cells and the high level of membrane-derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk. We investigated the number of MPs, their cellular origin and their procoagulant properties in beta-thalassaemia. Fresh whole blood was simultaneously stained for annexin V, cellular antigens and the known density beads. The procoagulant properties of these phosphatidylserine (PS)-bearing MPs were also measured by assessing the platelet factor-3-like activity in the blood. Flow cytometric results showed that splenectomised beta-thalassaemia/HbE patients had significantly higher levels of PS-bearing MPs than non-splenectomised beta-thalassaemia/HbE patients and normal individuals (P < 0.0001). There was a good correlation between PS-bearing MPs and PS-bearing platelets, reflecting the existence of chronic platelet activation in beta-thalassaemia/HbE patients (r(s) = 0.511, P < 0.001). The cellular origin of PS-bearing MPs showed mostly activated-platelet origin with adhesion (CD41a/CD62P/CD36). Moreover, the platelet procoagulant activity was higher in splenectomised beta-thalassaemia/HbE patients when compared with non-splenectomised (P < 0.05) and normal individuals (P < 0.01), and the amount correlated with PS-bearing MPs (rs = 0.560, P < 0.001). These findings suggest that MPs originate from activated platelets with a potential to aggravate thrombotic events when the numbers are excessive, as is commonly seen in splenectomised beta-thalassaemia/HbE patients.     

Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia

Paroxysmal nocturnal hemoglobinuria (PNH), frequently occurring during suppressed hematopoiesis including aplastic anemia (AA), is a clonal disorder associated with an increased incidence of thrombotic events. Complement-mediated hemolysis, impairment of the fibrinolytic system, or platelet activation are thought to be responsible for the associated thrombotic risk. We investigated here the elevation of membrane-derived procoagulant microparticles in the blood flow of such patients. Elevated levels of circulating microparticles were in fact detected in both de novo PNH patients and AA subjects with a PNH clone, but not in those with AA without a PNH clone. The cellular origin of the microparticles was determined in PNH samples; most stemmed from platelets. Glycophorin A+ particles were rarely detected. Therefore, platelet activation, resulting in the dissemination of procoagulant phospholipids in the blood flow, could be one of the main causes for the elevated thrombotic risk associated with PNH. These observations suggest that shed membrane particles can be considered a valuable biological parameter for the assessment of possible thrombotic complications in patients with PNH.     

MicroRNAs as potential regulators of platelet function and bleeding diatheses

Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.     

Inherited traits affecting platelet function

Inherited platelet disorders constitute a large group of diseases involving a wide range of genetic defects that can lead to bleeding symptoms of varying severity. They are associated with defects in surface membrane glycoproteins resulting in e.g. Bernard Soulier Syndrome and Glanzmann Thrombasthenia causing defects in platelet adhesion and aggregation, respectively, as well as in receptors for agonists (a.o. P2Y(12), TXA(2)) disrupting platelet signalling. Defects affecting platelet granules can be characterised by abnormalities of alpha-granules as in the Gray platelet syndrome or dense granules as in Hermansky-Pudlak and Chediak-Higashi syndromes, the latter two also altering other cytoplasmic organelles such as melanosomes and therefore not restricted to platelets. Finally, defects in proteins essential to signalling pathways (a.o. in Wiskott-Aldrich syndrome) or in platelet-derived procoagulant activity (Scott and Stormorken syndromes) also impair platelet function. For most of the above disorders mouse knockout models have been generated, that allowed to confirm the genotype-phenotype relationship and to further unravel the molecular causes of the disease and the mechanisms underlying primary haemostasis. More recently, interest has been growing in the effects of the more common polymorphisms that are found in the platelet glycoproteins as possible risk factors for thrombotic disorders. The new era of platelet genomics and proteomics will increase our knowledge on platelet disorders that will improve their diagnosis, but also will provide basis for new antithrombotic therapies.     

Role of FcRgamma and factor XIIIA in coated platelet formation

Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of alpha-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcRgamma and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the alphaIIbbeta3 activation-dependent antibody PE-JON/A. In FcRgamma-/- mice, only 10% of platelets became "coated" after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcRgamma-/- platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA-/- mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcRgamma, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.     

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.     

Clinical usefulness of a functional assay for the von Willebrand factor cleaving protease (ADAMTS 13) and its inhibitor in a patient with thrombotic thrombocytopenic purpura

Decreased von Willebrand factor cleaving protease activity (VWFCP, ADAMTS 13) leads to persistence of unusually large multimers of von Willebrand factor that bind to platelets, causing platelet aggregates, microangiopathic hemolysis, and thrombocytopenia in patients with thrombotic thrombocytopenic purpura (TTP). The clinical value of measuring ADAMTS 13 and its inhibitor is not fully defined; the case reported here illustrates the usefulness of the assay to help confirm the clinical diagnosis in a patient with other potential causes for thrombotic microangiopathy; the assay also helped in making treatment decisions. A patient with systemic lupus erythematosis (SLE) presented with fever and abdominal pain, thrombocytopenia, and anemia. Thrombotic microangiopathy was diagnosed by the appearance of schistocytes, decreasing platelet count, and evidence of hemolysis. ADAMTS 13 was decreased and an inhibitor was demonstrated in the patient's initial blood sample within 24 hr of admission. Plasma exchange was initiated, and serial assays showed increased ADAMTS 13 activity and decreased inhibitor after each plasma exchange; there was a rebound in inhibitor and a decrease in ADAMTS 13 activity prior to the next exchange that lessened over time. Increasing levels of protease activity correlated with clinical and laboratory improvement. Measurement of ADAMTS 13 activity and its inhibitor aided in the diagnosis of this complicated case of a patient with other potential causes for microangiopathic hemolysis. Subsequent levels correlated with the clinical course, and disappearance of the inhibitor indicated that long-term plasma exchange or other immunosuppressive treatment was not needed.     

Platelet prothrombin converting activity in hereditary disorders of platelet function

Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).     

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.     

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.     

血栓性疾病患者血小板相关组织因子水平及活性改变的意义

目的探讨血栓性疾病中血小板相关组织因子(TF)蛋白水平和活性的变化及其意义。方法采用Sepharose2B凝胶柱分离纯化血小板,ELISA法测定洗涤血小板破碎液TF蛋白含量;一期血浆凝固时间测定血小板相关TF促凝活性。结果正常人静息血小板TF蛋白(16·37±6·39)ng/L,未活化的血小板促凝活性为(0·89±0·26)U/ml,胶原活化后的血小板促凝活性为(2·27±0·24)U/ml,加入特异性TF单抗后活性为(1·39±0·28)U/ml,活化血小板的活性能被TF单抗显著抑制(P<0·001),证明活化后血小板的促凝活性主要来自TF。血栓性疾病中冠心病组、脑梗死组、糖尿病组血小板破碎液中TF蛋白水平分别为(20·71±8·78)、(23·83±8·03)、(22·12±8·24)ng/L,其对应的未经胶原处理的血小板促凝活性分别为(1·71±0·24)、(1·69±0·25)、(1·75±0·31)U/ml,加入TF单抗后分别为(1·43±0·25)、(1·39±0·25)、(1·40±0·29)U/ml,血栓性疾病患者血小板破碎液中TF蛋白水平及活性均较正常对照明显增高,其促凝活性可被TF单抗显著抑制(P<0·01)。结论血栓性疾病中血小板相关TF的变化可能参与了疾病过程中高凝状态的形成。     

New paradigms in thrombosis: novel mediators and biomarkers platelet RNA transfer

Platelets, anucleated cells with a central role in hemostasis and inflammation, contain messenger RNAs and microRNAs of unknown functionality and clinical relevance. Historically, platelet RNA was viewed as merely a remnant of platelet biogenesis; however, several studies now refute this assumption. Studies have shown that platelets can actively translate RNA to protein and that specific RNA profiles correlate with select human clinical phenotypes. These studies support a more fluid role for platelet RNA in platelet function and disease development. Our lab and others have recently studied the platelet’s unique ability to transfer RNA to recipient cells and the effect this transfer has on the recipient cells’ functions. This transfer may represent a previously unknown form of vascular cell communication and modulation. Unlike the well-characterized thrombotic properties of platelets, the nature and purpose of platelet RNA transfer has not been determined, partly due to limitations in techniques used to manipulate platelet RNA profiles. Defining the mechanism of RNA transfer and its role in the vascular system will allow for the better understanding of how platelets function in both their traditional thrombotic role and non-traditional functions, potentially having widespread implications in several fields.     

Plasma P-selectin is increased in thrombotic consumptive platelet disorders

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P- selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P- selectin may be a useful new marker for thrombotic diseases.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

A colorimetric, metabolic dye reduction assay detects highly activated platelets: application in the diagnosis of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is caused by antibodies, elicited in response to heparin anticoagulant therapy, which can cause extreme platelet activation and result in a highly procoagulant state. Most diagnostic tests for HIT antibodies measure in?vitro platelet activation by detecting aggregation or granule release responses. This study demonstrates that the high level of activation by HIT antibodies leads to a rapid breakdown of platelet metabolic activity. Resting or mildly activated platelets metabolize tetrazolium-based indicator dye to a dark colored product, in proportion to cell concentration and dye incubation time. Highly activated, procoagulant platelets resulting from incubation with HIT antibodies fail to metabolize dye and remain light in color. The loss of ability to metabolically reduce indicator dye provides a colorimetric endpoint that can be used in an in?vitro washed platelet activation assay to detect HIT antibodies. In a prospective evaluation, 145 diagnostic specimens were tested concurrently by both the colorimetric, dye reduction assay and the clinical laboratory standard, radiolabeled-serotonin release assay ((14)C-SRA). Results were in agreement for 96-100% of cases, depending on the chosen stringency of assay cut-off values. This study demonstrates that the metabolic dye reduction assay is comparable to the (14)C-SRA for HIT diagnosis. In addition, this novel assay may have even wider applicability, facilitating studies on the physiologic and clinical relevance of highly activated platelet populations.     

Thrombotic complications in essential thrombocythemia (ET): clinical facts and biochemical riddles

Hemostatic complications which can occur in the arterial or venous vasculature or in the microcirculation are the major causes of morbidity and mortality in patients with ET. In order to prevent these complications, often platelet reductive drugs are used. These agents are by themselves potentially toxic, i.e. may cause leukemia or cardiac side effects. In order to avoid these adverse effects, a better understanding of the mechanism of thrombus formation which is causative in ET is mandatory. Unfortunately, until now, no biomarkers have been identified which allow the estimation of the risk of thrombotic complications. Platelet number is not a good predictor per se since thrombotic complications can occur in some patients at low platelet numbers whereas others do not encounter a thrombosis even at very high platelet levels. On the other hand, lowering of the platelet count usually results in symptomatic improvement. In ET, morphological alterations of the megakaryocyte in the bone marrow and the circulating platelets are observed: megakaryocyte nuclei show a staghorn appearance, circulating platelets are characterized by anisocytosis and giant size. Functional studies indicate that these anatomically altered platelets function abnormally. When platelets are analyzed with a platelet function analyzer (PFA-100, which uses cartridges that measure how well a patient's platelets adhere and aggregate to form a platelet plug in the first phase of thrombus formation), in many patients with ET, closure time using collagen/ADP and collagen/epinephrine cartridges is prolonged. This seems paradoxical since these patients do not show an increased bleeding time. These results indicate that either receptors and/or consecutive signaling events are abnormal in ET platelets. Proteomic analysis of platelets of ET patients has revealed individual differences but not yet led to the identification of disease-specific proteins. Moreover, the search for alternative risk factors (factor V Leiden, prothrombin gene polymorphism, etc.) has not provided evidence for the contribution of these factors to the generation of the thrombotic risk in ET patients. In summary, despite intensive research over several decades, relatively little is known about the pathogenesis and risk factors for thrombosis in ET. I expect that this conference will contribute to the development of new strategies to identify patients at risk for hemostatic complications.     

Role of the coagulation system in tumor-cell-induced platelet aggregation and metastasis

The ability of tumor cells to initiate coagulation and subsequent platelet aggregation is believed to facilitate the metastatic process. The mechanism by which tumor cells initiate thrombotic alterations is unclear. We have purified a plasma membrane protein platelet aggregating activity/procoagulant activity (PAA/PCA) from several rodent tumors which initiates the coagulation of homologous plasma and aggregation of homologous platelets by a mechanism independent of factor VII. This protein does not possess any proteinase activity; however, its activity is dependent upon the presence of factor X. In addition, PAA/PCA requires reconstitution with phospholipid for expression of activity. These results suggest that tumor cells express a unique protein which possesses procoagulant activity resulting in thrombin generation. Thrombin is responsible for subsequent tumor-cell-induced platelet aggregation.     

The use of electron microscopy in the investigation of the ultrastructural morphology of immune thrombocytopenic purpura platelets

Ultrastructural studies have proven useful for the accurate identification and classification of certain lymphoid and hematopoietic disorders; however, the value of electron microscopy in the diagnosis and clinical management of platelet pathology is less well defined. Electron microscopy has been used to evaluate inherited platelet disorders. In these disorders, certain platelet structural defects can be characteristic. Recently, we investigated the ultrastructural morphology and immunogold localization of IgG in platelets from patients with idiopathic thrombocytopenic purpura (ITP). The accelerated platelet destruction of ITP is mediated by antiplatelet autoantibodies directed against platelet surface glycoproteins and by the functional capacity of the reticuloendothelial system. The basic dysregulation that occurs in these patients remains unexplained. Traditionally, laboratory investigation of ITP has focused on the development of serologic assays to measure the autoantibodies. As an alternative investigative approach, determination of the immunomorphologic characteristics of platelet-associated IgG (PAIgG) in ITP platelets may prove useful in the diagnosis or clinical management of patients with ITP. Our results showed that the ultrastructural morphology of ITP platelets is similar to that observed for normal platelets. No structural abnormalities are observed in ITP platelets. Immunogold labeling of IgG within alpha-granules of ITP platelets is significantly higher than that of normal platelets. Additionally, in some ITP platelets, immunogold labeling is also observed on the platelet surface and within channels of the open-cannalicular system. In comparison, immunogold labeling of these structures in normal platelets, is rare, or absent. In conclusion, electron microscopic studies should contribute to furthering our understanding of this common autoimmune disorder, and may provide possible biological explanations for the increased levels of PAIgG in platelets from patients with ITP.     

Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. In order to prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in eight patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (five females, three males) with a median age of 41.5 years (range, 24-53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5x109/L (range, 8-92). Three had a fatal outcome and five were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high-titer antibodies against platelet factor 4 (PF4) (optical density [OD] 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (Pselectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.