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The Journal of Clinical Investigation Jan 1977Human platelets washed and fixed in paraformaldehyde aggregate in the presence of the antibiotic ristocetin and normal plasma. This aggregation response is abolished...
Human platelets washed and fixed in paraformaldehyde aggregate in the presence of the antibiotic ristocetin and normal plasma. This aggregation response is abolished after digestion of the fixed platelets with chymotrypsin. Antisera to fixed washed platelets were produced in rabbits and absorbed with chymotrypsin-treated, fixed washed platelets. Monovalent Fab fragments obtained from the isolated gamma-globulin fractions of the antisera blocked ristocetin-induced aggregation of fixed washed platelets in buffer and normal platelets in platelet-rich plasma. By double-antibody immunoprecipitation, it was shown that the antibody which blocked the ristocetin reaction interacted with a platelet membrane surface protein of mol wt 155,000. The results suggest that the glycoprotein I complex on the surface of the human platelet mediates ristocetin-induced von Willebrand factor-dependent platelet aggregation.
Topics: Antibodies; Blood Platelets; Cell Membrane; Chymotrypsin; Factor VIII; Glycoproteins; Immune Sera; Platelet Aggregation; Ristocetin; von Willebrand Factor
PubMed: 299747
DOI: 10.1172/JCI108612 -
Journal of Thrombosis and Haemostasis :... Feb 2010The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades...
BACKGROUND
The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbalpha-VWF interaction.
OBJECTIVES
To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3zeta in apoptotic signaling.
METHODS
Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb-IX interacting with VWF by flow cytometry or western blotting.
RESULTS
Ristocetin-induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIbalpha antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with GPIbalpha truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3zeta-binding site in GPIbalpha.
CONCLUSIONS
This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3zeta with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.
Topics: 14-3-3 Proteins; Animals; Apoptosis; Binding Sites; Blood Platelets; CHO Cells; Cricetinae; Cricetulus; Gelsolin; Humans; Membrane Potential, Mitochondrial; Mutation; Phosphatidylserines; Platelet Activation; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Protein Structure, Tertiary; Ristocetin; Signal Transduction; Stress, Mechanical; Transfection; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; von Willebrand Factor
PubMed: 19840363
DOI: 10.1111/j.1538-7836.2009.03653.x -
Frontiers in Pharmacology 2019HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of...
Comparison of Effects of Anti-thrombin Aptamers HD1 and HD22 on Aggregation of Human Platelets, Thrombin Generation, Fibrin Formation, and Thrombus Formation Under Flow Conditions.
HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05-3 μM) and in washed platelets (WPs; 0.005-3 μM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 μM) and HD22 (0.5 μM), respectively. Neither HD1 nor HD22 (3 μM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 μM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 μM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 μM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC at 3 μM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 μM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 μM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 μM). values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure.
PubMed: 30842734
DOI: 10.3389/fphar.2019.00068 -
Scientific Reports Feb 2021The effect of conventional anti-platelet agents is limited in secondary stroke prevention, and their effects are blunted under high shear stress in the presence of...
The effect of conventional anti-platelet agents is limited in secondary stroke prevention, and their effects are blunted under high shear stress in the presence of increased levels of circulating von Willebrand factor (VWF). VWF is critically involved in thrombus formation at sites of stenotic extracranial/intracranial arteries. A third generation anti-VWF aptamer (BT200) has been generated which could be useful for secondary stroke prevention. To characterize the effects of BT200 in blood of patients with large artery atherosclerosis stroke (LAA). Blood samples were obtained from 33 patients with acute stroke or transient ischemic attack to measure inhibition of VWF activity and VWF-dependent platelet function. Patients who received clopidogrel or dual antiplatelet therapy did not differ in VWF dependent platelet function tests from aspirin treated patients. Of 18 patients receiving clopidogrel with or without aspirin, only 3 had a prolonged collagen adenosine diphosphate closure time, and none of the patients had ristocetin induced aggregation in the target range. BT200 concentration-dependently reduced median VWF activity from 178 to < 3%, ristocetin induced platelet aggregation from 40U to < 10U and prolonged collagen adenosine diphosphate closure times from 93 s to > 300 s. Baseline VWF activity correlated (r = 0.86, p < 0.001) with concentrations needed to reduce VWF activity to < 20% of normal, indicating that BT200 acts in a target concentration-dependent manner. Together with a long half-life supporting once weekly administration, the safety and tolerability observed in an ongoing phase I trial, and the existence of a reversal agent, BT200 is an interesting drug candidate.
Topics: Aged; Aptamers, Peptide; Aspirin; Blood Platelets; Collagen; Female; Humans; Intracranial Arteriosclerosis; Ischemic Attack, Transient; Male; Platelet Aggregation; Stroke; Thrombosis; von Willebrand Factor
PubMed: 33542410
DOI: 10.1038/s41598-021-82747-7 -
Blood Aug 1983It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that... (Comparative Study)
Comparative Study
It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that low concentrations of thrombin also cause platelet membrane receptors to become available for FVIII/vWF. As a consequence, the suspicion has been raised that thrombin provides similar or equivalent activity in vivo to that observed for ristocetin under in vitro conditions. In this study, we quantitated the extent to which thrombin promotes the binding of FVII/vWF to platelets and determined whether or not this interaction initiates or complements platelet aggregation. With ristocetin present, the amount of 125I-FVIII/vWF that became platelet-bound correlated closely with the onset, rate, and extent of platelet aggregation. In contrast, at every thrombin concentration tested, the amount of 125I-FVIII/vWF that specifically bound to platelets was about 6% of that observed with ristocetin. Significantly, FVIII/vWF did not augment the rate of aggregation of platelets in response to thrombin or initiate platelet aggregation when subaggregating doses of thrombin were used. These observations indicate that the minimal association that occurs between FVIII/vWF and the platelet membrane in the presence of thrombin does not correlate with platelet aggregation and therefore is not analogous to the effects of ristocetin. Whether the low level of binding relates to another process, such as platelet-endothelial interactions, remains unknown.
Topics: Blood Platelets; Cell Membrane; Humans; In Vitro Techniques; Platelet Aggregation; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Ristocetin; Thrombin
PubMed: 6307433
DOI: No ID Found -
Xenotransplantation 2012Platelet activation/aggregation plays a key role in the dysregulation of coagulation and the development of thrombotic microangiopathy in nonhuman primate recipients of... (Comparative Study)
Comparative Study
INTRODUCTION
Platelet activation/aggregation plays a key role in the dysregulation of coagulation and the development of thrombotic microangiopathy in nonhuman primate recipients of pig xenografts. As a preliminary to the study of anti-platelet therapy in vitro and in vivo, the present study aimed to compare platelet aggregation in whole blood from humans, baboons, and cynomolgus monkeys.
METHODS
Using "Chrono-log" technology (two-sample four-channel Chrono-log Whole Blood Aggregometer), we studied aggregation of platelets in healthy humans (n = 8), baboons (n = 5), and monkeys (n = 8). Whole blood (WB) samples were collected, and platelet aggregation was assessed using three different volumes of blood (1, 0.5, and 0.25 ml). Platelet activation was induced using collagen (at 3 and 5 μg/ml), ristocetin (at 0.5 and 1.0 mg/ml), adenosine diphosphate (ADP; at 10, 20, and 40 μm), or thrombin (at 1 and 5 IU/ml). Inhibition of agonist-induced platelet aggregation by heparin and low molecular weight heparin (LMWH) (at 1, 10, and 100 IU/ml) was evaluated.
RESULTS
Mean platelet counts were 222.1, 263.2, and 276.1 (×10(3) /μl) in humans, baboons, and monkeys, respectively. In all three species, platelet aggregation was induced by collagen, ristocetin, ADP, or thrombin in a dose-dependent manner. A blood volume of 0.5 ml provided the most consistent results with all agonists in all three species. Dilution studies indicated that there was a significant positive correlation between platelet count and percent aggregation of platelets (P < 0.05). Collagen (3 and 5 μg/ml), ADP (10, 20, and 40 μm), and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in humans than in baboons. ADP (20 and 40 μm) and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in monkeys than in baboons. There was no species difference with ristocetin (0.5 or 1.0 mg/ml). In all species, thrombin (1 or 5 IU) induced greater platelet aggregation than any of the other reagents. Heparin at 1 IU/ml and LMWH at 10 IU/ml in all species almost completely abrogated thrombin-induced platelet aggregation. Heparin at 100 IU/ml effectively inhibited platelet aggregation induced by collagen, but only partially inhibited aggregation induced by ADP or ristocetin. LMWH only partially inhibited aggregation induced by collagen, ristocetin, and ADP.
CONCLUSIONS
The "Chrono-log" technology proved to be a reliable method of evaluating platelet activation and aggregation in vitro in primates. Species differences may play a role in platelet aggregation, with the monkey being more comparable to the human than the baboon, although overall trends were similar. In all species, thrombin induced greater platelet aggregation than other agonists. Even a concentration of heparin of 1 IU/ml, which is probably the maximal concentration that is clinically-applicable, prevented platelet aggregation induced by thrombin, but was less effective in preventing aggregation induced by collagen, ADP, or, particularly, ristocetin.
Topics: Adenosine Diphosphate; Animals; Collagen; Heparin; Heparin, Low-Molecular-Weight; Humans; In Vitro Techniques; Macaca fascicularis; Papio anubis; Platelet Aggregation; Platelet Aggregation Inhibitors; Ristocetin; Species Specificity; Thrombin; Transplantation, Heterologous
PubMed: 22909136
DOI: 10.1111/j.1399-3089.2012.00712.x -
Research and Practice in Thrombosis and... Oct 2022Von Willebrand disease (VWD) is a common inherited bleeding disorder, however the diagnosis can be complicated by a subjective bleeding history and issues with some...
BACKGROUND
Von Willebrand disease (VWD) is a common inherited bleeding disorder, however the diagnosis can be complicated by a subjective bleeding history and issues with some current von Willebrand factor (VWF) laboratory assays.
OBJECTIVES
In the Zimmerman Program, we sought to determine how often a type 1 diagnosis was based on a single low VWF ristocetin cofactor (VWF:RCo) level resulting from the common genetic variant p.D1472H or an isolated assay issue, if that low value was corroborated by the VWF glycoprotein-IbM (VWF:GPIbM) assay, and if retesting confirmed original levels.
METHODS
New patients being evaluated for bleeding were consented. Analysis included sequencing, bleeding scores, and comparisons of local VWF antigen (VWF:Ag) and VWF:RCo to central VWF:Ag and VWF:GPIbM.
RESULTS
A total of 18% of VWD subjects had a low local VWF:RCo, but normal VWF:Ag and normal central testing including VWF:GPIbM. Seventy percent of the low VWF:RCo cohort had no pathogenic variants; however, 33% carried p.D1472H. Low VWF:RCo subjects with follow-up local testing within 2 years showed those with p.D1472H continued to have low VWF:RCo and VWF:RCo/VWF:Ag ratio with normal VWF:GPIbM. Subjects without p.D1472H had an increase mean VWF:RCo, resulting in 59% with normal levels on repeat testing.
CONCLUSIONS
The diagnosis of VWD based on a single low VWF:RCo but normal VWF:Ag, was often attributed to p.D1472H or variability in VWF:RCo that was eliminated with VWF:GPIbM. Our study suggests that using VWF:RCo alone for diagnostic purposes may be insufficient while repeat VWF:RCo or VWF:GPIbM testing can be valuable in establishing a VWD diagnosis.
PubMed: 36381287
DOI: 10.1002/rth2.12807 -
Artificial Organs Jul 2016Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS)...
Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.
Topics: Adult; Blood Platelets; Female; Hemorrhage; Humans; Male; Middle Aged; Platelet Activation; Platelet Aggregation; Platelet Membrane Glycoproteins; Stress, Mechanical; Thrombosis; Young Adult; von Willebrand Factor
PubMed: 26582038
DOI: 10.1111/aor.12606 -
World Journal of Gastroenterology Apr 2017To investigate serum omentin and vaspin levels in cirrhotic patients; and to assess the relationship of these levels with hemostatic parameters, metabolic abnormalities,... (Observational Study)
Observational Study
AIM
To investigate serum omentin and vaspin levels in cirrhotic patients; and to assess the relationship of these levels with hemostatic parameters, metabolic abnormalities, cirrhosis severity and etiology.
METHODS
Fifty-one cirrhotic patients (17 with portal vein thrombosis) were analyzed. Serum omentin and vaspin levels were measured with commercially available direct enzyme-linked immunosorbent assays (ELISAs). To assess platelet activity, the following tests were performed using a MULTIPLATEPLATELET FUNCTION ANALYZER: (1) an ADP-induced platelet activation test; (2) a cyclooxygenase dependent aggregation test (ASPI test); (3) a von Willebrand factor and glycoprotein Ib-dependent aggregation (using ristocetin) test (RISTO test); and (4) a test for thrombin receptor-activating peptide-6 induced activation of the thrombin receptor, which is sensitive to IIb/IIIa receptor antagonists.
RESULTS
Omentin, but not vaspin, serum concentrations were significantly decreased in patients with portal vein thrombosis (PVT) ( = 0.01). Prothrombin levels were significantly increased in patients with PVT ( = 0.01). The thrombin receptor activating peptide (TRAP) test results were significantly lower in the PVT group ( = 0.03). No significant differences in adipokines serum levels were found regarding the etiology or severity of liver cirrhosis assessed according to the Child-Pugh or Model of End-Stage Liver Disease (MELD) scores. There was a significant increase in the TRAP ( = 0.03), ASPI ( = 0.001) and RISTO high-test ( = 0.02) results in patients with lower MELD scores. Serum omentin and vaspin levels were significantly down-regulated in patients without insulin resistance ( = 0.03, = 0.02, respectively). A positive relationship between omentin and vaspin levels were found both when all of the patients were analyzed ( = 0.41, = 0.01) and among those with PVT ( = 0.94, < 0.001).
CONCLUSION
Serum omentin levels are increased in patients without PVT. Cirrhosis origin and grade do not affect omentin and vaspin levels. The analyzed adipokines do not influence platelet activity.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; GPI-Linked Proteins; Humans; Insulin Resistance; Lectins; Liver Cirrhosis; Male; Middle Aged; Platelet Aggregation; Platelet Function Tests; Portal Vein; Prothrombin; Serpins; Severity of Illness Index; Venous Thrombosis
PubMed: 28465646
DOI: 10.3748/wjg.v23.i14.2613 -
Blood Dec 1997Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into...
Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.
Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Binding Sites; Blood Platelets; COS Cells; Calcium; Cells, Cultured; Epitopes; Flow Cytometry; Humans; Macaca fascicularis; Molecular Sequence Data; Oligopeptides; Papio; Peptides; Platelet Activation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Swine; von Willebrand Factor
PubMed: 9373253
DOI: No ID Found