Erik Westein

A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation.

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t½α = 13 min) and a slow secondary phase (t½β = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D′-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D′-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D′-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D′-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.

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.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.

Cysteine-mutations in von Willebrand factor associated with increased clearance

Summary.  Background: von Willebrand disease (VWD) is a bleeding disorder caused by the decrease of functional von Willebrand factor (VWF). Low levels of VWF can result from decreased synthesis, impaired secretion, increased clearance or combinations thereof. Several mutations lead to impaired synthesis or secretion of VWF, however, little is known about the survival of VWF in the circulation. Objectives: To evaluate the effect of several VWF mutations on VWF clearance. Patients/methods: The effect of three cysteine‐mutations (C1130F, C1149R or C2671Y) on the in vivo survival of VWF was studied in patients carrying these mutations and in a VWF‐deficient mice model. Results: In patients carrying these mutations, we observed increased propeptide/mature VWF ratios and rapid disappearance of VWF from the circulation after desmopressin treatment. Detailed analysis of in vivo clearance of recombinant VWF in a VWF‐deficient mice model revealed a fourfold increased clearance rate of the mutants. The mutations C1130F, C1149R and C2671Y are each associated with reduced survival of VWF in the circulation. Detailed analysis of the recombinant mutant VWF demonstrated that increased clearance was not due to increased proteolysis by ADAMTS‐13. We did not identify functional or structural characteristics that the mutant proteins have in common and could be associated with the phenomenon of increased clearance. Conclusions: Cysteine‐mutations in VWF may result in reduced in vivo survival. The observation that various mutations are associated with increased in vivo clearance may have major implications for the therapeutic strategies that rely on the rise of endogenous VWF after desmopressin administration.

Evidence of Platelet Activation at Medically Used Hypothermia and Mechanistic Data Indicating ADP as a Key Mediator and Therapeutic Target

Objective—Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Methods and Results—Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl3-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P2Y12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. Conclusion—The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

Platelet Recruitment to the Inflamed Glomerulus Occurs via an αIIbβ3/GPVI-Dependent Pathway

Recruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbalpha but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)-dependent mechanism, and inhibition of alpha(IIb)beta(3), fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the alpha(IIb)beta(3)/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment.

Towards Effective and Safe Thrombolysis and Thromboprophylaxis: Preclinical Testing of a Novel Antibody-Targeted Recombinant Plasminogen Activator Directed Against Activated Platelets

Rationale: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. Objective: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. Methods and Results: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet–integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5–scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline ( P <0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5–scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5–scuPA as compared with the same dose of a mutated, nontargeting scFv–scuPA or vehicle. The ability of scFvSCE5–scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. Conclusions: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications. # Novelty and Significance {#article-title-48}Rationale: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. Objective: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. Methods and Results: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet–integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5–scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5–scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5–scuPA as compared with the same dose of a mutated, nontargeting scFv–scuPA or vehicle. The ability of scFvSCE5–scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. Conclusions: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Monitoring in vitro thrombus formation with novel microfluidic devices

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices.

Multifunctional Thrombin-Activatable Polymer Capsules for Specific Targeting to Activated Platelets.

Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis.