scholarly journals Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions

2016 ◽  
Vol 104 (6) ◽  
pp. 661-668
Author(s):  
Yasunori Matsunari ◽  
Mitsuhiko Sugimoto ◽  
Masaaki Doi ◽  
Hideto Matsui ◽  
Masahiko Kawaguchi
Keyword(s):  
Blood Flow ◽  
Tissue Factor ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Functional Characterization ◽  
Flow Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3626-3632 ◽  
Author(s):  
Barbara Plaimauer ◽  
Klaus Zimmermann ◽  
Dirk Völkel ◽  
Gerhard Antoine ◽  
Randolf Kerschbaumer ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Functional Activity ◽  
Functional Characterization ◽  
Eukaryotic Expression ◽  
Gene Encoding ◽  
Hek 293 ◽  
293 Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designatedADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.

scholarly journals Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 345-353 ◽  
Author(s):  
RR Hantgan ◽  
G Hindriks ◽  
RG Taylor ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Rates ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

scholarly journals COVID-19 and Acute Coronary Syndromes: From Pathophysiology to Clinical Perspectives

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Luca Esposito ◽  
Francesco Paolo Cancro ◽  
Angelo Silverio ◽  
Marco Di Maio ◽  
Patrizia Iannece ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Tissue Factor ◽  
Von Willebrand Factor ◽  
Acute Coronary Syndromes ◽  
Oxygen Supply ◽  
Thrombus Formation ◽  
Cellular Damage ◽  
Coronary Syndromes ◽  
Von Willebrand ◽  
Willebrand Factor

Acute coronary syndromes (ACS) are frequently reported in patients with coronavirus disease 2019 (COVID-19) and may impact patient clinical course and mortality. Although the underlying pathogenesis remains unclear, several potential mechanisms have been hypothesized, including oxygen supply/demand imbalance, direct viral cellular damage, systemic inflammatory response with cytokine-mediated injury, microvascular thrombosis, and endothelial dysfunction. The severe hypoxic state, combined with other conditions frequently reported in COVID-19, namely sepsis, tachyarrhythmias, anemia, hypotension, and shock, can induce a myocardial damage due to the mismatch between oxygen supply and demand and results in type 2 myocardial infarction (MI). In addition, COVID-19 promotes atherosclerotic plaque instability and thrombus formation and may precipitate type 1 MI. Patients with severe disease often show decrease in platelets count, higher levels of d-dimer, ultralarge von Willebrand factor multimers, tissue factor, and prolongation of prothrombin time, which reflects a prothrombotic state. An endothelial dysfunction has been described as a consequence of the direct viral effects and of the hyperinflammatory environment. The expression of tissue factor, von Willebrand factor, thromboxane, and plasminogen activator inhibitor-1 promotes the prothrombotic status. In addition, endothelial cells generate superoxide anions, with enhanced local oxidative stress, and endothelin-1, which affects the vasodilator/vasoconstrictor balance and platelet aggregation. The optimal management of COVID-19 patients is a challenge both for logistic and clinical reasons. A deeper understanding of ACS pathophysiology may yield novel research insights and therapeutic perspectives in higher cardiovascular risk subjects with COVID-19.

scholarly journals Synthetic Partially Reduced Human Neutrophil Peptide (HNP)-1 Inhibits Platelet Adhesion and Aggregation on Von Willebrand Factor-Collagen Surfaces Under Arterial Shear Stress through a Disulfide Bond Reduction Mechanism

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3722-3722
Author(s):  
Jenny K. McDaniel ◽  
Khalil Bdeir ◽  
Douglas B. Cines ◽  
X. Long Zheng
Keyword(s):  
Mass Spectrometry ◽  
Disulfide Bond ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Human Neutrophil ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Reduction Mechanism ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: Human neutrophil peptides (HNPs) are small cationic proteins primarily released from activated and degranulated neutrophils. HNPs have antimicrobial activity against diverse bacteria, viruses, fungi, and parasites. Additionally, HNPs exhibit prothrombotic properties by enhancing platelet aggregation and fibrin formation and by inhibiting proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13. However, the role of HNPs in thrombus formation under more physiological conditions (i.e. under flow) has not been determined. Objective: To investigate the effects of HNPs on platelet adhesion/aggregation on VWF/collagen surfaces under arterial shears. Design/Method: Whole blood was obtained from C57/BL6 wild type and Adamts13-/-mice, anticoagulated with a potent thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) and prostaglandin-E1 (PGE1), and platelets were labeled with FITC anti-mouse CD41 IgG. After incubation with varying concentrations of native HNPs and synthetic partially reduced HNP1 (sHNP1) for 30 minutes, the whole blood samples were perfused through a fibrillar collagen-coated surface in a microfluidic system at 100 dyne/cm² for 180 seconds. The rate and extent of accumulation of fluorescein-labeled platelets were determined under an inverted fluorescent microscope at 4-second intervals. The images were analyzed off-line with Montage to evaluate the area of platelet coverage over time. This process was repeated with the addition of N-ethylmaleimide (NEM) alone or NEM-treated sHNP1 to the samples to probe the effect of free cysteine residues. In addition, samples of native HNPs and sHNP1 incubated with NEM were analyzed via LC-mass spectrometry for NEM incorporation. Results: Purified native HNPs at final concentrations of 15 μM and 30 μM exhibited no or little effect on the adhesion and aggregation of murine platelets on VWF/collagen surfaces under arterial shears (100 dyne/cm2). Surprisingly, sHNP1 at the same concentrations (15 and 30 μM) dramatically reduced the rate and surface coverage of platelets from WT (Fig. 1A) and, more profoundly, from Adamts13-/- mice (Fig. 1B) on VWF/collagen surfaces under the same conditions. This inhibitory activity of sHNP1 was abolished upon pretreatment with NEM, which reacts with free thiols (-SH) (not shown). Aliphatic HNP1 with all 6 cysteine residues chemically modified also did not inhibit the adhesion and aggregation of murine platelets on VWF/collagen surfaces under shear (not shown). Analysis of samples by LC-mass spectrometry confirmed the NEM-labeling of free thiols present in sHNP1, but not in native HNPs. Conclusion: These results suggest that high concentrations of locally released native HNPs may be required to inhibit ADAMTS13 activity in vivo. However, the findings from this study indicate that HNPs differentially affect thrombus formation depending on how its redox state is modified by its biological milieu. Somewhat unexpectedly, synthetic and partially reduced HNP1 may be a potent antithrombotic agent by reducing platelet interactions with VWF under arterial shear via a disulfide bond reduction mechanism. Disclosures Zheng: Alexion: Research Funding; Ablynx: Consultancy.

scholarly journals Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4413-4424 ◽  
Author(s):  
Masaaki Moroi ◽  
Stephanie M. Jung ◽  
Shosaku Nomura ◽  
Sadayoshi Sekiguchi ◽  
Antonio Ordinas ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Initial Reaction ◽  
Adhesion Assay ◽  
Flow Conditions ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

scholarly journals Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 345-353 ◽  
Author(s):  
RR Hantgan ◽  
G Hindriks ◽  
RG Taylor ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Rates ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

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