Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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Platelet Activation and Biofilm Formation by Aerococcus urinae, an Endocarditis-Causing Pathogen

Infection and Immunity ◽

10.1128/iai.00469-10 ◽

2010 ◽

Vol 78 (10) ◽

pp. 4268-4275 ◽

Cited By ~ 29

Author(s):

Oonagh Shannon ◽

Matthias Mörgelin ◽

Magnus Rasmussen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Biofilm Formation ◽

Platelet Rich Plasma ◽

Protein Structures ◽

Prostaglandin E ◽

Bacterial Surface ◽

Fibrinogen Receptor ◽

Aerococcus Urinae

ABSTRACT The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

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ARHGEF12 Is Essential for Human Megakaryocyte Differentiation and Plays Critical Roles in Platelet Function

Blood ◽

10.1182/blood.v124.21.341.341 ◽

2014 ◽

Vol 124 (21) ◽

pp. 341-341

Author(s):

Siying Zou ◽

Alexandra M Teixeira ◽

Chad D Sanada ◽

Ping-xia Zhang ◽

Diane Krause

Keyword(s):

Platelet Aggregation ◽

Mouse Model ◽

Western Blot ◽

Platelet Activation ◽

Platelet Function ◽

Blot Analysis ◽

Western Blot Analysis ◽

Thrombin Receptors ◽

Platelet Functions

Abstract Megakaryocytopoiesis, the process by which hematopoietic stem cells develop into mature megakaryocytes (MK), and thrombopoiesis, platelet production/release, are critical for blood homeostasis. We tested the hypothesis that the Rho guanine exchange factor, ARHGEF12 (also known as LARG), is critical for MK differentiation and platelet functions based on the following: 1) ARHGEF12 is part of a recurrent translocation with MLL in acute myeloid leukemia. 2) Both published microarray datasets and deep-sequencing data from our lab on primary human CD34+ cells differentiating into MKs show that ARHGEF12 expression goes up dramatically during MK differentiation. 3) ARHGEF12 is one of the most highly expressed guanine exchange factors in platelets. 4) ARHGEF12 forms a complex with G proteins and stimulates Rho-dependent signals. It is known that platelet activation can be initiated by extracellular stimuli working through G protein-coupled receptors and Rho signaling, suggesting that ARHGEF12 may function in platelet activation. 5) Mice with KO of RhoA (a known ARHGEF12 substrate) in the MK-lineage have macrothrombocytopenia and defective platelet activation. To test this hypothesis, we used ARHGEF12 shRNA mediated KD and an ARHGEF12 specific pharmacological inhibitor (Y16) in both murine and human primary cells, and characterized a LARG KO mouse model for MK and platelet phenotypes, and found: ARHGEF12 is differentially upregulated during MK differentiation and is enriched in platelets Using quantitative RT-PCR and western blot analysis at different timepoints of primary FACSorted Mk progenitors induced to differentiate into mature MK in vitro, ARHGEF12 RNA and protein expression increases during MK differentiation in both the murine and human systems. Also western blot analysis of murine platelet rich plasma shows that ARHGEF12 protein is highly expressed in platelets. ARHGEF12 is essential for human MK differentiation To test the function of ARHGEF12 in Mk differentiation, we used lentiviral shRNA to knockdown ARHGEF12 in FACSorted primary human Mk progenitors from mobilized peripheral blood differentiated in vitro to MK. The results show that ARHGEF12 knockdown blocks MK polyploidization (not shown) and maturation (Fig. A). This was confirmed using a published ARHGEF12 inhibitor (Y16) in the differentiation culture of human MK progenitors, in which there was a dose-dependent block in MK differentiation (Fig. B). These data suggested that ARHGEF12 is essential for human MK differentiation. We researched the function of ARHGEF12 in the murine system using a constitutive ARHGEF12 knockout mouse model. The mice have enlarged platelets (p=0.07) and a decreased platelet count (p=0.01). However, the knockout mice have normal BM cellularity with no change in megakaryocyte number or ploidy, suggesting that ARHGEF12 is dispensable for murine MK differentiation in vivo. ARHGEF12 is essential for platelet function in both the murine and human systems: To test whether ARGEF12 functions in platelet activation, we compared WT versus KO platelet activation in vitro. We tested activation in response to ADP, U46619 (Thromboxane), ADP+U46619, and Thrombin. KO plateelts have significantly reduced activation in response to U46619 and thrombin, with no effects on ADP-induced activation. Analogous studies using the ARHGEF12 inhibitor (Y16) on WT platelets revealed supportive evidence. Lastly, we tested ARHGEF12 function in human platelet aggregation using the Y16 compound. Consistent with the murine data, Y16 blocked platelet aggregation in response to both U46619 and Thrombin. Taken together, these data strongly suggest that ARHGEF12 is essential for platelet function and acts downstream of the Thromboxane and Thrombin receptors. In summary, we found that ARHGEF12 is differentially up-regulated in MK differentiation both in human and in mouse system,. It plays a critical role in human Mk differentiation but is dispensable in murine MK differentiation, and ARHGEF12 is critical for platelet functions in both human and mouse systems, potentially acting downstream of Thromboxane and Thrombin receptors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Potent in Vitro and in Vivo Inhibitors of Platelet Aggregation Based Upon the Arg-Gly-Asp Sequence of Fibrinogen. (Aminobenzamidino)succinyl (ABAS) Series of Orally Active Fibrinogen Receptor Antagonists

Journal of Medicinal Chemistry ◽

10.1021/jm00013a014 ◽

1995 ◽

Vol 38 (13) ◽

pp. 2378-2394 ◽

Cited By ~ 67

Author(s):

Jeffery A. Zablocki ◽

Joseph G. Rico ◽

Robert B. Garland ◽

Thomas E. Rogers ◽

Kenneth Williams ◽

...

Keyword(s):

Platelet Aggregation ◽

Receptor Antagonists ◽

Fibrinogen Receptor ◽

Orally Active

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Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation

Thrombosis and Haemostasis ◽

10.1160/th16-11-0856 ◽

2017 ◽

Vol 117 (08) ◽

pp. 1651-1659 ◽

Cited By ~ 6

Author(s):

Ann-Katrin Mojica Muñoz ◽

Janina Jamasbi ◽

Kerstin Uhland ◽

Heidrun Degen ◽

Götz Münch ◽

...

Keyword(s):

Platelet Aggregation ◽

Antiplatelet Therapy ◽

Platelet Adhesion ◽

Dual Antiplatelet Therapy ◽

Platelet Inhibition ◽

Antiplatelet Drug ◽

Fibrinogen Receptor ◽

Static Conditions ◽

P2y12 Antagonist

SummaryThe efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y12 antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53%, and increased platelet inhibition by ASA (51%) and ticagrelor (64%) to 66% and 80%, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57%, and significantly increased platelet inhibition by ASA (28%) and ticagrelor (47%) to about 81% each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93%). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti-fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81%) and stable (89%) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual antiplatelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/IIIa inhibitors could be harmful.Note: The review process for this manuscript was fully handled by Gregory Y. H. Lip, Editor in Chief.Supplementary Material to this article is available at www.thrombosis-online.com.

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Molecular Structure and Biological Activity of NPP-4, An Endothelial Cell Surface Pyrophosphatase/ Phosphodiesterase That Stimulates Platelet Aggregation and Secretion Via Liberation of ADP Upon Hydrolysis of Diadenosine Triphosphate

Blood ◽

10.1182/blood.v118.21.701.701 ◽

2011 ◽

Vol 118 (21) ◽

pp. 701-701

Author(s):

Deborah L. Ornstein ◽

Ronald Albright ◽

William C. Chang ◽

Donna Robert ◽

Wenxiang Cao ◽

...

Keyword(s):

Endothelial Cell ◽

Platelet Aggregation ◽

Extracellular Space ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Dense Granule ◽

Structural Basis ◽

Diadenosine Triphosphate ◽

Granule Release

Abstract Abstract 701 Diadenosine triphosphate (Ap3A) is a component of platelet dense granules, and is released into the extracellular space during the second wave of platelet aggregation following platelet activation by ADP and other agonists. Ap3A has long been thought to stimulate platelet aggregation after release into the extracellular space by providing a prolonged source of ADP via hydrolysis by a slow extracellular enzyme present in human plasma. Here, we identify NPP-4, a member of the nucleotide pyrophosphatase/phosphodiesterase enzyme family present on endothelial cell surfaces, as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion at nanomolar concentrations. We performed lumiaggregometry with citrated platelet-rich plasma in the presence of Ap3A with or without increasing nanomolar concentrations of NPP-4. In these experiments we determined that Ap3A alone in concentrations up to 80 uM initiated a primary wave of platelet aggregation that was followed by rapid disaggregation. In the presence of nanomolar concentrations of NPP-4, however, the primary and secondary waves of platelet aggregation and dense granule release are strongly induced in an enzyme concentration-dependent fashion. In contrast, mutant NPP-4, catalytically inactivated by an active site threonine to alanine mutation, had no effect on platelet aggregation and dense granule release under identical conditions. In order to clarify the structural basis of the enzymatic mechanism, we determined the high-resolution structure of NPP4 in the presence and absence of the enzymatic product, AMP. In aggregate, our studies define the biological, enzymatic, and molecular basis for NPP-4 and Ap3A activity in platelet aggregation and secretion in vitro and suggest that NPP-4 may play a role in hemostasis in vivo by augmenting platelet aggregation and release of granule contents at the site of vascular injury. With these studies we have thus established the molecular foundation for the rational development of a new class of therapeutics capable of modulating vascular thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Abstract 450: Phosphorylation Of Platelet αIIbβ3 Is Crucial For Arterial Thrombosis In Vivo And Microparticle Generation

Circulation ◽

10.1161/circ.116.suppl_16.ii_75-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Manojkumar Valiyaveettil ◽

Weiyi Feng ◽

Ganapati Mahabaleshwar ◽

David Phillips ◽

Tatiana Byzova ◽

...

Keyword(s):

Platelet Aggregation ◽

Tyrosine Phosphorylation ◽

Arterial Thrombosis ◽

Thrombus Formation ◽

Annexin V ◽

Vessel Occlusion ◽

Occlusion Time ◽

Fibrinogen Receptor

Functional activity of platelet fibrinogen receptor αIIbβ3 is crucial for hemostasis and thrombosis. The process of αIIbβ3 activation in platelet aggregation is tightly regulated. It has been previously shown that β3 subunit of the complex undergo tyrosine phosphorylation, which, in turn, is believed to control recruitment of several intracellular adaptors. Mutations of Tyr747/ Tyr759 within the cytoplasmic domain of αIIbβ3 (DiYF substitution) were found to result in reversible platelet aggregation. To assess whether αIIbβ3 tyrosine phosphorylation is critical for arterial thrombosis, we utilized intravital microscopy to monitor thrombus formation in vivo in WT and DiYF mice. Compared to WT, DiYF mice exhibited delayed platelet adhesion and reduced rate of thrombus formation at the initial stages of thrombosis. Likewise, isolated DiYF platelets exhibited reduced adhesion to collagen under in vitro sheer conditions compared to WT. The progression phase of thrombosis in vivo was similar in WT and DiYF mice. The most dramatic difference was observed at the final phase of thrombus formation since it took 3-times longer for blood vessels in DiYF mice to occlude compared to WT. To specifically address the role if β3 phosphorylation in platelet αIIbβ3 vs αvβ3 on leukocytes and vascular cells, we transfused labeled WT and DiYF platelets into irradiated WT mice with low blood cells and platelet counts. It was found that transfusion of DiYF but not WT platelets resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion time in vivo. Similar differences were observed in tail bleeding test. Importantly, we have found that the lack of β3 phosphorylation impaired an ability of platelets to generate microparticles in response to activation, an event believed to be critical for the final stages of thrombosis. When stimulated with thrombin and PMA, DiYF platelets shed ~50% less Annexin V-positive microparticles as compared to WT platelets. Thus, β3 tyrosine phosphorylation of αIIbβ3 in platelets is crucial for the microparticles generation by activated platelets and the overall process of arterial thrombosis in vivo.

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Effect of Temperature on ADP-Induced Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647758 ◽

1973 ◽

Vol 29 (01) ◽

pp. 183-189

Author(s):

C. A Praga ◽

E. M Pogliani

Keyword(s):

Platelet Aggregation ◽

Incubation Period ◽

Room Temperature ◽

Important Variable ◽

Practical Significance ◽

Effect Of Temperature ◽

Induce Platelet Aggregation ◽

Cuvette Holder ◽

Exact Temperature

SummaryTemperature represents a very important variable in ADP-induced platelet aggregation.When low doses of ADP ( < 1 (μM) are used to induce platelet aggregation, the length of the incubation period of PRP in the cuvette holder of the aggregometer, thermostatted at 37° C, is very critical. Samples of the same PRP previously kept at room temperature, were incubated for increasing periods of time in the cuvette of the aggregometer before adding ADP, and a significant decrease of aggregation, proportional to the length of incubation, was observed. Stirring of the PRP during the incubation period made these changes more evident.To measure the exact temperature of the PRP during incubation in the aggre- gometer, a thermocouple device was used. While the temperature of the cuvette holder was stable at 37° C, the PRP temperature itself increased exponentially, taking about ten minutes from the beginning of the incubation to reach the value of 37° C. The above results have a practical significance in the reproducibility of the platelet aggregation test in vitro and acquire particular value when the effect of inhibitors of ADP induced platelet aggregation is studied.Experiments carried out with three anti-aggregating agents (acetyl salicyclic acid, dipyridamole and metergoline) have shown that the incubation conditions which influence both the effect of the drugs on platelets and the ADP breakdown in plasma must be strictly controlled.

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Evidence for an Increased Generation of Prostacyclin in the Microvasculature and an Impairment of the Platelet α-Granule Release in Chronic Renal Failure

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647030 ◽

1988 ◽

Vol 60 (02) ◽

pp. 205-208 ◽

Cited By ~ 21

Author(s):

Paul A Kyrle ◽

Felix Stockenhuber ◽

Brigitte Brenner ◽

Heinz Gössinger ◽

Christian Korninger ◽

...

Keyword(s):

Renal Failure ◽

Chronic Renal Failure ◽

Blood Clotting ◽

Normal Subjects ◽

Chronic Uraemia ◽

Wall Interaction ◽

End Stage ◽

Granule Release

SummaryThe formation of prostacyclin (PGI2) and thromboxane A2 and the release of beta-thromboglobulin (beta-TG) at the site of platelet-vessel wall interaction, i.e. in blood emerging from a standardized injury of the micro vasculature made to determine bleeding time, was studied in patients with end-stage chronic renal failure undergoing regular haemodialysis and in normal subjects. In the uraemic patients, levels of 6-keto-prostaglandin F1α (6-keto-PGF1α) were 1.3-fold to 6.3-fold higher than the corresponding values in the control subjects indicating an increased PGI2 formation in chronic uraemia. Formation of thromboxane B2 (TxB2) at the site of plug formation in vivo and during whole blood clotting in vitro was similar in the uraemic subjects and in the normals excluding a major defect in platelet prostaglandin metabolism in chronic renal failure. Significantly smaller amounts of beta-TG were found in blood obtained from the site of vascular injury as well as after in vitro blood clotting in patients with chronic renal failure indicating an impairment of the a-granule release in chronic uraemia. We therefore conclude that the haemorrhagic diathesis commonly seen in patients with chronic renal failure is - at least partially - due to an acquired defect of the platelet a-granule release and an increased generation of PGI2 in the micro vasculature.

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Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

Author(s):

Hiroh Yamazaki ◽

Itsuro Kobayashi ◽

Tadahiro Sano ◽

Takio Shimamoto

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

The Other ◽

Endothelial Surface ◽

Important Interaction ◽

Blood Coagulability ◽

The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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