Marijke J.E. Kuijpers

Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner

Rupture of a vulnerable atherosclerotic plaque causes thrombus formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in thrombus formation. How the altered hemodynamics around a plaque promote pathological thrombus formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and thrombus formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60–80% channel occlusion over a range of input wall shear rates. The poststenotic thrombus formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent thrombus formation in the setting of atherosclerosis.

Segregation of Platelet Aggregatory and Procoagulant Microdomains in Thrombus Formation. Regulation by Transient Integrin Activation

Objective—Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results—High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIIbβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3. Conclusions—These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.

Adhesion of human and mouse platelets to collagen under shear: a unifying model

There is presently confusion as to the roles of α2β1 and GPVI in supporting platelet adhesion and aggregate formation on collagen at intermediate/high shear. Recent studies have reported essential, partial, or dispensable roles for either receptor in supporting these events, and the possibility that there may be fundamental differences between their roles in human and mouse platelets has been proposed. Further, the recent recognition that Src family tyrosine kinases contribute to signaling by α2β1 and other adhesive receptors, in addition to GPVI, has added to this debate. The present study compares the roles of α2β1, GPVI, and Src‐dependent kinases in supporting adhesion and aggregation in human and mouse platelets in whole blood using blocking antibodies, mutant mice, and a novel inhibitor of Src kinases, PD0173952, which is effective in plasma. The results demonstrate that the fundamental processes of adhesion and aggregate formation are conserved in mice and human platelets and that two mechanisms of stable adhesion and activation on collagen exist. These can be distinguished by the contributions of GPVI and α2β1, with GPVI‐mediated platelet activation either preceding or following integrin‐mediated adhesion. The relative contribution of each pathway depends on environmental conditions and may also reflect platelet heterogeneity. These observations form the basis of a unifying two‐state model of platelet adhesion and aggregate formation on collagen that is conserved between human and mouse platelets.

Principal Role of Glycoprotein VI in α2β1 and αIIbβ3 Activation During Collagen-Induced Thrombus Formation

Objective—High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of &agr;2&bgr;1 and &agr;IIb&bgr;3 integrin activation during thrombus formation on collagen. Methods and Results—Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of &agr;2&bgr;1 and &agr;IIb&bgr;3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin &agr;2&bgr;1 and &agr;IIb&bgr;3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and &agr;2&bgr;1, indicating that the integrin can support platelet–collagen binding in the absence of its activation. Blockade or absence of GPIb&agr; only moderately influenced integrin activation and adhesion unless GPVI was inhibited. Conclusions—GPVI- and autocrine-released ADP induce affinity changes of &agr;2&bgr;1 and &agr;IIb&bgr;3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet–collagen interactions and thereby collagen-induced platelet activation.

Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen‐bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso‐occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin α2β1, is essential in priming platelet‐collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen‐dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin β1 reduces the GPVI‐evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in Gαq or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen‐induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin α2β1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction.

The Glycoprotein VI-Phospholipase Cγ2 Signaling Pathway Controls Thrombus Formation Induced by Collagen and Tissue Factor In Vitro and In Vivo

Objective—Both collagen and tissue factor can be initiating factors in thrombus formation. We investigated the signaling pathway of collagen-induced platelet activation in interaction with tissue factor–triggered coagulation during the thrombus-forming process. Methods and Results—In murine blood flowing over collagen, platelet exposure of phosphatidylserine and procoagulant activity, but not adhesion, completely relied on each of the following signaling modules: glycoprotein VI (GPVI), FcR &ggr;-chain, Src kinases, adaptor protein LAT, and phospholipase C&ggr;2 (PLC&ggr;2). On flow in the presence of tissue factor, these signaling components were essential for platelet aggregation and greatly enhanced fibrin clot formation. Collagen-stimulated thrombin generation relied on the presence and activity of GPVI, FcR &ggr;-chain, Src kinase, LAT, and PLC&ggr;2. The physiological importance of this GPVI pathway was shown in a FeCl3-induced in vivo murine thrombosis model. In both venules and arterioles, signaling through GPVI, FcR &ggr;-chain, and Src kinases enhanced the formation of phosphatidylserine-exposing and fibrin-rich thrombi. Conclusions—The GPVI-PLC&ggr;2 activation pathway regulates collagen-dependent coagulation in venous and arterial thrombus formation.

Complementary roles of glycoprotein VI and α2β1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen‐bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso‐occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin α2β1, is essential in priming platelet‐collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen‐dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin β1 reduces the GPVI‐evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in Gαq or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen‐induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin α2β1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Factor XII Regulates the Pathological Process of Thrombus Formation on Ruptured Plaques

Objective— Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor–factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo. Approach and Results— Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe −/− mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas. Conclusions— The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor–FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.

Imaging Collagen in Intact Viable Healthy and Atherosclerotic Arteries Using Fluorescently Labeled CNA35 and Two-Photon Laser Scanning Microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E−/− mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E−/− mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.