From Discovery of Snake Venom Disintegrins to A Safer Therapeutic Antithrombotic Agent

Snake venoms affect blood coagulation and platelet function in diverse ways. Some venom components inhibit platelet function, while other components induce platelet aggregation. Among the platelet aggregation inhibitors, disintegrins have been recognized as unique and potentially valuable tools for examining cell–matrix and cell–cell interactions and for the development of antithrombotic and antiangiogenic agents according to their anti-adhesive and anti-migration effect on tumor cells and antiangiogenesis activities. Disintegrins represent a family of low molecular weight, cysteine-rich, Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing polypeptides, which inhibit fibrinogen binding to integrin αIIbβ3 (i.e., platelet glycoprotein IIb/IIIa), as well as ligand binding to integrins αvβ3, and α5β1 expressed on cells (i.e., fibroblasts, tumor cells, and endothelial cells). This review focuses on the current efforts attained from studies using disintegrins as a tool in the field of arterial thrombosis, angiogenesis, inflammation, and tumor metastasis, and briefly describes their potential therapeutic applications and side effects in integrin-related diseases. Additionally, novel R(K)GD-containing disintegrin TMV-7 mutants are being designed as safer antithrombotics without causing thrombocytopenia and bleeding.

Download Full-text

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649933 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1316-1322 ◽

Cited By ~ 13

Author(s):

Mary Ann McLane ◽

Jagadeesh Gabbeta ◽

A Koneti Rao ◽

Lucia Beviglia ◽

Robert A Lazarus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Sequence Similarity ◽

Platelet Aggregation Inhibitors ◽

Antiplatelet Drug ◽

Rgd Sequence ◽

Fibrinogen Receptor ◽

Activated Platelets

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.

Download Full-text

Partial Inhibition of Platelet Aggregation and Fibrinogen Binding by a Murine Monoclonal Antibody to GPIIIa: Requirement for Antibody Bivalency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648991 ◽

1994 ◽

Vol 72 (06) ◽

pp. 964-972 ◽

Cited By ~ 16

Author(s):

Jeffery L Kutok ◽

Barry S Coller

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Murine Monoclonal Antibody ◽

Platelet Glycoprotein ◽

Igg Antibody ◽

Surface Receptors ◽

Partial Inhibition ◽

Fibrinogen Binding ◽

Igg Binding ◽

Agonist Concentration

SummaryWe produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) Ilia. 7H2-IgG and 7H2-F(ab’)2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, ∼40,000 7H2-IgG antibody molecules bind per platelet. In contrast, ∼80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s).

Download Full-text

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650238 ◽

1996 ◽

Vol 75 (01) ◽

pp. 168-174 ◽

Cited By ~ 13

Author(s):

Shigeru Tokita ◽

Morio Arai ◽

Naomasa Yamamoto ◽

Yasuhiro Katagiri ◽

Kenjiro Tanoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Heparan Sulfate ◽

Disulfide Bonds ◽

Dextran Sulfate ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent Fashion ◽

Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

Download Full-text

Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽

10.1182/blood.v68.3.783.bloodjournal683783 ◽

1986 ◽

Vol 68 (3) ◽

pp. 783-786 ◽

Cited By ~ 1

Author(s):

BS Coller ◽

JD Folts ◽

LE Scudder ◽

SR Smith

Keyword(s):

Monoclonal Antibody ◽

Animal Model ◽

Platelet Aggregation ◽

Ex Vivo ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Antithrombotic Effect ◽

Antithrombotic Agent ◽

Antithrombotic Effects ◽

Platelet Thrombus

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

Download Full-text

Impaired function of platelet membrane glycoprotein IIb-IIIa in end-stage renal disease.

Journal of the American Society of Nephrology ◽

10.1681/asn.v5136 ◽

1994 ◽

Vol 5 (1) ◽

pp. 36-46

Author(s):

M P Gawaz ◽

G Dobos ◽

M Späth ◽

P Schollmeyer ◽

H J Gurland ◽

...

Keyword(s):

Renal Failure ◽

Chronic Renal Failure ◽

Platelet Aggregation ◽

Platelet Function ◽

Uremic Toxin ◽

Bleeding Tendency ◽

Fibrinogen Receptor ◽

Fibrinogen Binding ◽

Uremic Patients ◽

Stage Renal Disease

Impaired platelet function and a bleeding tendency are well-recognized complications of chronic renal failure. Because the fibrinogen receptor GPIIb-IIIa plays a central role in platelet aggregation and adhesion to the subendothelium, it was reasoned that a defect in this receptor may underlie the impaired platelet function in uremia. To test this hypothesis, the function of this receptor in the platelets of 11 uremic patients was studied. Aggregation studies were performed with flow cytometric techniques with anti-GPIIb-IIIa conformation-specific monoclonal antibodies (mAb) (anti-LIBS1 and anti-PMI-1). Antifibrinogen and antithrombospondin mAb were used to characterize fibrinogen binding to GPIIb-IIIa and the release of alpha-granules, respectively. Platelets from patients with chronic renal failure showed significantly decreased binding of conformation-dependent anti-LIBS1 mAb after ADP, phorbol myristate acetate, or RGD-peptide stimulation compared with normal controls, suggesting a defect related to the ability of the fibrinogen receptor to undergo a conformational change. Moreover, antifibrinogen and antithrombospondin binding to activated platelets were reduced in uremic patients, implying impairment of both ligand-binding and alpha-granule release. Hemodialysis partially restored GPIIb-IIIa function, which may account for the observed effects of this therapy in restoring platelet aggregation. These findings indicate that platelets of patients with chronic renal failure reveal an aggregation defect at least partially due to an intrinsic GPIIb-IIIa dysfunction and the presence of a putative uremic toxin that inhibits fibrinogen binding to GPIIb-IIIa.

Download Full-text

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽

10.1182/blood.v91.10.3792.3792_3792_3799 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3792-3799 ◽

Cited By ~ 3

Author(s):

Hilde Depraetere ◽

Nadine Ajzenberg ◽

Jean-Pierre Girma ◽

Catherine Lacombe ◽

Dominique Meyer ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Platelet Glycoprotein ◽

Rotational Viscometer ◽

Induce Platelet Aggregation ◽

Static Conditions ◽

Von Willebrand ◽

Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Download Full-text

IMPORTANCE OF LARGE VON WILLEBRAND FACTOR (vWF) MULTIMERS IN vWF INTERACTION WITH PLATELET GLYCOPROTEIN IIb/IIIa

10.1055/s-0038-1644096 ◽

1987 ◽

Author(s):

M Yamamoto ◽

Y Ando ◽

K Watanabe ◽

H Iri ◽

Y Araki ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Platelet Glycoprotein ◽

Induce Platelet Aggregation ◽

Maximum Extent ◽

Physiological Relevance ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor

Recently it has been reported that, in addition to binding to glycoprotein (GP) lb, vWF also interacts with GPIIb/IIIa, although the physiological relevance of this interaction is not completeley clear. In this paper, we have investigated the role of different size of vWF multimers in vWF-mediated platelet aggregation. Different size of vWF multimers were purified from human plasma through Sephacryl S-1000 column according to the method of Fowler et al. Fractions were analysed by SDS-agarose gel electrophoresis by the method of Ruggeri et al. When each fraction was examined for ristocetin cofactor activity (RCo), only larger multimers exhibited significant RCo. The maximum extent of ristocetin-induced platelet agglutination by larger multimers (10 μg/ml) was 80%, while that of intermediate and lower multimers at the same concentration was 20% and 0%, respectively. Each fraction was then added to washed platelet suspensions in the presence of 10 μM ADP and 0.3 mM CaCl2. Only larger multimers induced platelet aggregation, while intermediate and lower multimers failed to induce platelet aggregation. The maximum extent of aggregation in the presence of larger multimers (10 μg/ml) was 70% of that in the presence of fibrinogen instead. Similar experiments were peformed using platelet-rich plasma from a patient with afibrinogenemia in stead of washed normal platelets. ADP caused significant aggregation only when purified vWF larger multimers or fibrinogen was added. This vWF-mediated aggregation was completely inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM).Our results indicate that larger multimers of vWF play major roles in vWF interaction with GPIIb/IIIa.

Download Full-text

A Critical Role for Membrane Sulfatide in Platelet Aggregation.

Blood ◽

10.1182/blood.v104.11.626.626 ◽

2004 ◽

Vol 104 (11) ◽

pp. 626-626

Author(s):

Prasenjit Guchhait ◽

Corie Shrimpton ◽

Kochi Honke ◽

Perumal Thiagarajan ◽

Jose A. Lopez

Keyword(s):

Platelet Aggregation ◽

Lipid Rafts ◽

Platelet Function ◽

Critical Role ◽

Membrane Microdomains ◽

Systemic Lupus Erythematosis ◽

Single Chain ◽

Induce Platelet Aggregation ◽

Adhesive Interactions

Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

Download Full-text

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽

10.1182/blood.v120.21.2168.2168 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2168-2168

Author(s):

Lu Wang ◽

Yi Wu ◽

Junsong Zhou ◽

Syed S. Ahmad ◽

Bulent Mutus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Protein Disulfide Isomerase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Platelet Surface ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Astilbe chinensis Modulates Platelet Function via Impaired MAPK and PLCγ2 Expression

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/3835021 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Bo-Ra Jeon ◽

Muhammad Irfan ◽

Seung Eun Lee ◽

Jeong Hoon Lee ◽

Man Hee Rhee

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Plant Extract ◽

Signaling Molecules ◽

Calcium Mobilization ◽

Therapeutic Effects ◽

Western Blots ◽

Downstream Signaling ◽

Fibrinogen Binding ◽

Granule Secretion

Background. Platelets play major role in maintaining hemostasis while hyperactivation of platelets may lead to arterial thrombosis. Natural products and ethnomedicine have been shown to reduce the risk of cardiovascular diseases (CVDs). Astilbe chinensis is a perennial herb found in China, Korea, Russia, and Japan, which is also known for its medicinal effects, and has been used in Korean traditional medicine to treat inflammation, cancer, chronic bronchitis, and headache. We hypothesized that given herbal plant exhibits pharmacological activities against CVDs, and we specifically explored their effects on platelet function. Methodology. Platelet aggregation was evaluated using standard light-transmission aggregometry. Intracellular calcium mobilization was assessed using Fura-2/AM, and granule secretion (ATP release) was measured in a luminometer. Fibrinogen binding to integrin αIIbβ3 was assessed using flow cytometry. Phosphorylation of mitogen-activated protein kinase (MAPK) signaling molecules and activation of the phosphoinositide 3-kinase (PI3K)/Akt were assessed using western blots, and further, glycoprotein VI (GPVI) signaling components were studied using immunoprecipitation. Key Results. A. chinensis extracts potently and significantly inhibited platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin αIIbβ3. Moreover, it significantly inhibited MAPK phosphorylation and expression of GPVI downstream signaling molecules. Conclusion. A. chinensis extract inhibited platelet aggregation and granule secretion and attenuated GPVI downstream signaling, indicating the potential therapeutic effects of this plant extract on the cardiovascular system and platelet function. We suggest that given plant extract may be a potent candidate to treat platelet-related CVDs and to be used as antiplatelet agent.

Download Full-text