Inhibition Of Clot Retraction Research Articles

Antiplatelet mechanism of a subtilisin-like serine protease from Solanum tuberosum (StSBTc-3)

The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bβ3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bβ3 preventing the interaction between α2bβ3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.

Elevated Pre- and Postoperative ROTEM™ Clot Lysis Indices Indicate Reduced Clot Retraction and Increased Mortality in Patients Undergoing Liver Transplantation.

Background: The ROTEM™ clot lysis index, describing the decrease in firmness of a clot with time, predicts mortality in various settings. The variability of the clot lysis index in surgical procedures and the involved pathophysiological mechanisms are unknown. We therefore compared pre- and postoperative clot lysis indices in liver transplantation (LTX) procedures, determined the eventual association with mortality, and investigated the mechanisms underlying decreased clot lysis index using inhibitors of fibrinolysis and clot retraction, respectively. Methods: In this retrospective cohort study, data on pre- and post-transplant ROTEM™ findings as obtained with EXTEM (tissue factor activation), INTEM (intrinsic system activation), FIBTEM (extrinsic system activation and inhibition of clot retraction), APTEM (extrinsic system activation and fibrinolysis inhibition), conventional laboratory coagulation tests, blood loss, transfusion of blood products, and outcome were registered. Results: Pre-transplant clot lysis indices showed a broad distribution ranging from 75% to 99% independent of the activator used (EXTEM, INTEM). During the surgical procedure, median clot lysis index values markedly increased from 92% to 97% (EXTEM) and 93% to 98% (INTEM), respectively (p < 0.0001 each). Aprotinin had no effect on either pre- or postsurgical clot lysis indices. Inhibition of platelet clot retraction with cytochalasin D (FIBTEM) markedly increased the preoperative clot lysis index. High pre- and post-transplantation clot lysis indices were associated with increased mortality irrespective of the activator used (EXTEM, INTEM) and the inhibition of fibrinolysis (APTEM). Inhibition of clot retraction (FIBTEM) abolished the association of clot lysis index with mortality in both pre- and post-transplantation samples. Conclusion: Both pre- and postoperative ROTEM™ clot lysis indices predict mortality in patients following liver transplantation. Inhibitor experiments reveal that the clot lysis index is not an indicator of fibrinolysis, but indicates platelet clot retraction. The marked increase of clot lysis index during liver transplantation is caused by a decrease in clot retraction with eventual consequences for clot stability, retraction of wound margins, and reperfusion of vessels in case of thrombosis.

Novel contact-kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke.

Ischemic stroke is a leading cause of death and disability worldwide. Increasing evidence indicates that ischemic stroke is a thromboinflammatory disease in which the contact-kinin pathway has a central role by activating pro-coagulant and pro-inflammatory processes. The blocking of distinct members of the contact-kinin pathway is a promising strategy to control ischemic stroke. Here, a plasma kallikrein and active FXII (FXIIa) inhibitor (sylvestin, contained 43 amino acids, with a molecular weight of 4790.4Da) was first identified from forest leeches (Haemadipsa sylvestris). Testing revealed that sylvestin prolonged activated partial thromboplastin time without affecting prothrombin time. Thromboelastography and clot retraction assays further showed that it extended clotting time in whole blood and inhibited clot retraction in platelet-rich plasma. In addition, sylvestin prevented thrombosis in vivo in FeCl3-induced arterial and carrageenan-induced tail thrombosis models. The potential role of sylvestin in ischemic stroke was evaluated by transient and permanent middle cerebral artery occlusion models. Sylvestin administration profoundly protected mice from ischemic stroke by counteracting intracerebral thrombosis and inflammation. Importantly, sylvestin showed no signs of bleeding tendency. The present study identifies sylvestin is a promising contact-kinin pathway inhibitor that can proffer profound protection from ischemic stroke without increased risk of bleeding.

Clot Retraction: Cellular Mechanisms and Inhibitors, Measuring Methods, and Clinical Implications.

Platelets have important functions in hemostasis. Best investigated is the aggregation of platelets for primary hemostasis and their role as the surface for coagulation leading to fibrin- and clot-formation. Importantly, the function of platelets does not end with clot formation. Instead, platelets are responsible for clot retraction through the concerted action of the activated αIIbβ3 receptors on the surface of filopodia and the platelet’s contractile apparatus binding and pulling at the fibrin strands. Meanwhile, the signal transduction events leading to clot retraction have been investigated thoroughly, and several targets to inhibit clot retraction have been demonstrated. Clot retraction is a physiologically important mechanism allowing: (1) the close contact of platelets in primary hemostasis, easing platelet aggregation and intercellular communication, (2) the reduction of wound size, (3) the compaction of red blood cells to a polyhedrocyte infection-barrier, and (4) reperfusion in case of thrombosis. Several methods have been developed to measure clot retraction that have been based on either the measurement of clot volume or platelet forces. Concerning the importance of clot retraction in inborn diseases, the failure of clot retraction in Glanzmann thrombasthenia is characterized by a bleeding phenotype. Concerning acquired diseases, altered clot retraction has been demonstrated in patients with coronary heart disease, stroke, bronchial asthma, uremia, lupus erythematodes, and other diseases. However, more studies on the diagnostic and prognostic value of clot retraction with methods that have to be standardized are necessary.

Dominant role of αIIbβ3 in platelet interactions with cross-linked fibrin fragment D-dimer

AbstractAlthough much is known about the interaction of fibrinogen with αIIbβ3, much less is known about the interaction of platelets with cross-linked fibrin. Fibrinogen residue Lys406 plays a vital role in the interaction of fibrinogen with αIIbβ3, but because it participates in fibrin cross-linking, it is not available for interacting with αIIbβ3. We studied the adhesion of platelets and HEK cells expressing normal and constitutively active αIIbβ3 to both immobilized fibrinogen and D-dimer, a proteolytic fragment of cross-linked fibrin, as well as platelet-mediated clot retraction. Nonactivated platelets and HEK cells expressing normal αIIbβ3 adhered to fibrinogen but not D-dimer, whereas activated platelets as well as HEK cells expressing activated αIIbβ3 both bound to D-dimer. Small-molecule antagonists of the αIIbβ3 RGD (Arg-Gly-Asp) binding pocket inhibited adhesion to D-dimer, and an Asp119Ala mutation that disrupts the β3 metal ion–dependent adhesion site inhibited αIIbβ3-mediated adhesion to D-dimer. D-dimer and a polyclonal antibody against D-dimer inhibited clot retraction. The monoclonal antibody (mAb) 10E5, directed at αIIb and a potent inhibitor of platelet interactions with fibrinogen, did not inhibit the interaction of activated platelets with D-dimer or clot retraction, whereas the mAb 7E3, directed at β3, inhibited both phenomena. We conclude that activated, but not nonactivated, αIIbβ3 mediates interactions between platelets and D-dimer, and by extrapolation, to cross-linked fibrin. Although the interaction of αIIbβ3 with D-dimer differs from that with fibrinogen, it probably involves contributions from regions on β3 that are close to, or that are affected by, changes in the RGD binding pocket.

The antithrombotic effect of caffeic acid is associated with a cAMP-dependent pathway and clot retraction in human platelets

Caffeic acid (CA) is a polyphenol widely distributed in the plant kingdom. Studies have shown CA possesses antithrombotic activity in mouse cerebral arterioles in vivo and inhibits platelet aggregation in vitro. However, little is known regarding the effects of CA on platelet-mediated clot retraction. We investigated the effects of CA on platelet activation and clot retraction in response to thrombin. CA inhibited thrombin-induced platelet aggregation, calcium mobilization, adenosine 1,4,5-tri-phosphate (ATP) release, P-selectin expression and fibrinogen binding to integrin αIIbβ3 activation without inducing any cytotoxic effect, and inhibited the phosphorylations of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) in thrombin-stimulated platelets. In addition, CA enhanced cyclic adenosine monophosphate (cAMP) generation, which led to the phosphorylations of vasodilator-stimulated phosphoprotein (VASP) and inositol trisphosphate (IP3) receptor, and reduced clot retraction without any anticoagulation effect. Dipyridamole, a phosphodiesterase 3 (PDE3) inhibitor, reduced clot retraction, which suggested CA-mediated cAMP generation is the main signaling pathway responsible for its inhibition of clot retraction. Taken together, the findings of the present study suggest that CA may have potential as a therapeutic for the prevention of thrombotic disorders.

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbβ3, TXA2, and cAMP levels

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbβ3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule.In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbβ3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25–100 μM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbβ3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbβ3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.

Platelet bioenergetics and its implications in disease

Bioenergetics drives energy production for mechanical processes that are critical to platelet functions, e.g., adhesion, spreading, aggregation, clot formation and contraction. Storage and pathological conditions e.g., stroke, sepsis, diabetes etc., alters platelet energetics and function; however, the specific mechanisms governing these alterations are not clear. It is not well understood which metabolic fuels can be used and which are required. We are investigating the roles of different metabolic pathways in the energy consuming process of clot retraction. Using a spectrum of techniques from in vitro platelet functional assays to biochemical methods, whole animal models, and measurement of bioenergetic functions of mitochondria we are elucidating platelet bioenergetics and its implications in disease. We developed a simple method for measuring clot contraction kinetics using time lapse imaging. Our system is efficient, high throughput and is favorable to study platelet bioenergetics of clot formation and retraction. We hypothesize that, upon activation with thrombin, fuel use can be highly plastic, depending on the fuels available. We found that glycolysis and oxidative phosphorylation can both contribute to provide energy for platelet function. Inhibitors of glycolysis and oxidative phosphorylation using 2‐Deoxyglucose and oligomycin inhibited clot retraction showing that both these processes are important.We have also begun to examine bioenergetics in platelets from diabetics. Platelets from type2 diabetes patients have been shown to have abnormal mitochondrial membrane potential, membrane fluidity, ATP content, and oxidative stress. However, the impact of diabetes on platelet bioenergetics is less clear. We have developed a mouse model to probe the role of mitochondria and oxidative phosphorylation in platelet function. TFAM (Mitochondrial Transcription factor A) regulates mitochondria DNA copy number and transcription of mitochondrial DNA encoded proteins. The regulation of mitochondrial DNA is necessary for mitochondrial biogenesis. We have selectively deleted TFAM in the megakaryocyte lineage using the Platelet factor 4 promoter driven Cre‐Lox system. This model will be very useful, and it supports our in vitro studies with metabolic inhibitors in understanding the role of mitochondria and oxidative phosphorylation in platelet function in platelet bioenergetics contributing to the thrombosisSupport or Funding InformationSupported by HL56652, HL138179 and VA Merit AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the ‘RGD pocket’ formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, but not unactivated, Ps adhere well to immobilized 'D98;' 2. cells expressing normal αIIbβ3 could bind to: a) fbg in the absence of EDTA, but not in the presence of EDTA, b) D100 in the absence or presence of EDTA, c) 'D98' in the presence, but not the absence, of EDTA; 3. cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3, adhere well to immobilized 'D98.' These data support there being two separate mechanisms of binding, one involving the interaction of γ-404-411 (in fbg and D100) with the αIIbβ3 RGD binding pocket, and another involving a site on D100 and 'D98' (but cryptic in fbg) that interacts with a site on αIIbβ3 exposed by receptor activation or induced by EDTA.We have now studied adhesion of αIIbβ3 to the cross-linked fibrin fragment D-dimer (D-d). We predicted it to be similar to the interaction of ‘D98‘ with αIIbβ3 because γ-K406, which is important in the binding of fbg to αIIb, participates in the antiparallel reciprocal factor XIIIa-mediated transglutaminase crosslinks between K406 and Q398 on two different γ-chains, and so is unlikely to be able to interact with the RGD pocket.Methods: D-d, purified from cross-linked fibrin after plasmin digestion, was obtained from Haematologic Technologies and found to contain a dominant band with the expected Mr and immunoblot pattern using antibodies to the individual fbg chains. Fibrinogen, 'D98,' and HEK293 cells expressing normal and mutated αIIbβ3 were prepared as we previously described (Zafar et al., Blood Advances 2017), and the adhesion assays were conducted as described in that paper. Clot retraction was performed by adding BSA, 'D98,' or D-d (200 µg/ml) to washed Ps (2 X 108/ml) + thrombin (0.2 U/ml) in a glass aggregometer cuvette.Results: αIIbβ3-mediated adhesion to immobilized D-d (10 µg/ml coating concentration) was similar to adhesion to 'D98' in that 1. Unactivated P adhered well to fbg, but poorly to 'D98' and D-d (n=4); 2. Activated Ps (10 µM thrombin receptor activating peptide) adhere ~2-fold better than unactivated Ps to fbg, but ~10-fold better to 'D98' and D-d (p<0.001 for unactivated vs activated Ps with both 'D98' and D-d; n=4); 3. HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) adhere well to fibrinogen, but not 'D98' and D-d; 4. HEK293 cells expressing the constitutively active aIIbF992A/F993Aβ3 receptor [αIIb(FF)β3-HEK] bound well to fbg, 'D98, and D-d (p<0.001 for both 'D98' and D-d compared to normal αIIbβ3; n=5); 5. EDTA treatment of Ps results in loss of adhesion to fbg, but significantly enhanced adhesion to 'D98' and D-d (p<0.05 for each ligand with and without EDTA; n=4); 6. EDTA treatment of αIIbβ3-HEK results in loss of adhesion to fbg, but significantly enhanced adhesion to both 'D98' and D-d (p<0.01 for each ligand with and without EDTA; n=5); 7. Adhesion of αIIb(FF)β3-HEK to D-d is like adhesion to 'D98' in being inhibitable by mAbs 7E3 and 10E5, eptifibatide, tirofiban, and RUC-4 (p<0.01 for all); n=4); 8. Adhesion of αIIb(FF)β3-HEK to D-d is similar to adhesion to 'D98' in being inhibitable by mutating β3 D119 to A in the MIDAS (p<0.01; n=6); 9. EDTA-induced adhesion of HEK-αIIbβ3 to both 'D98' and D-d is not inhibitable by mutating β3 D119 to A. 10. 'D98' and D-d inhibited clot retraction, but BSA did not.Conclusion: We conclude that cross-linked fibrin fragment D-dimer, like fibrinogen fragment ‘D98‘, interacts with activated αIIbβ3 via sites other than the γ-404-411 peptide, and that this interaction contributes to clot retraction. DisclosuresColler:Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation.

The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

Chitosan-platelet-rich plasma implants for tissue repair - in vitro and in vivo characteristics

Event Abstract Back to Event Chitosan-platelet-rich plasma implants for tissue repair - in vitro and in vivo characteristics Gabrielle Depres-Tremblay1, Anik Chevrier2, Nicolas Tran-Khan2, Monica Nelea2 and Michael Buschmann1, 2 1 Polytechnique de Montreal, Biomedical engineering institute, Canada 2 Polytechnique de Montreal, Chemical engineering department, Canada Introduction: Freeze-dried chitosan formulations can be solubilised in platelet-rich plasma (PRP) to produce injectable mixtures that coagulate to form implants that resist platelet-mediated clot retraction and are resident for at least 2 weeks in vivo[1],[2]. The purpose of this study was to 1) investigate the mechanisms by which clot retraction is inhibited in chitosan-PRP hybrid clots, 2) to investigate release of platelet-derived growth factors from chitosan-PRP hybrid clots in culture medium, and 3) to inject freeze-dried chitosan-PRP formulations subcutaneously in rabbits to assess long-term implant retention and biodegradability. Materials and Methods: Freeze-dried chitosan cakes were prepared using 0.56% or 1% w/v chitosan (80% DDA and Mn 40kDa), 1% or 6% w/v trehalose concentration as lyoprotectant and 42.2mM calcium chloride (CaCl2) as clot activator. Cakes were solubilised in PRP obtained from a human donor and dispensed in either 1) Glass tubes for confocal and scanning electron microscopy imaging or 2) 24-well plates to monitor release of PDGF-AB over 7 days. Formulations containing 1% (w/v) chitosan of Mn 40kDa and different DDA (80%, 85%, 90%), 1% (w/v) trehalose and 42.2mM CaCl2 were solubilised with autologous PRP and injected subcutaneously in the back of five NZW rabbits (150µL/implant) to investigate implant retention and biodegradability at 2 weeks; 4 weeks and 6 weeks. Results: Platelet aggregates were smaller in chitosan-PRP hybrid clots compared to PRP only controls and increasing trehalose concentration further decreased the size of platelet aggregates (Fig 1 left). Platelets, cells and fibrin fibers were coated with chitosan in chitosan-PRP hybrid clots, while fibrin fibers were readily visible in PRP only controls (Fig 1 right). PDGF-AB release profiles were similar for the different chitosan-PRP hybrid clots and for PRP controls, but all were higher than whole blood alone (Fig 2 left). In vivo, PRP controls were completely degraded by 2 weeks, while all chitosan-PRP formulations tested (80%, 85% & 90% DDA) were retained up to 6 weeks post-implantation, where they induced cell recruitment of host-derived cells (Fig 2 right). Discussion: Chitosan inhibited platelet aggregation, partially through the physical mechanism of coating the cells and fibrin fibers in the hybrid clots. Increasing trehalose concentration further decreased the size of platelet aggregates through a currently unknown mechanism. Strong platelet aggregation is required for platelet-mediated clot retraction, so this is consistent with our previous results (1). PDGF-AB release was similar in chitosan-PRP hybrid clots and in PRP controls, which is unsurprising considering that PDGF-AB has an isoelectric point of 9.8 and solubilised chitosan-PRP formulations have a pH between 6.8-7.1. Chitosan-PRP hybrids resided up to 6 weeks post-implantation in vivo, while PRP controls were quickly degraded, suggesting that prolonged residency of chitosan itself leads to sustained recruitment of host cells. Conclusion: Chitosan and trehalose decreased the size of platelet aggregates, which is consistent with their inhibition of clot retraction. Chitosan-PRP does not provide sustained release of PDGF-AB but may do so for other growth factors with different charge properties. Prolonged residency of chitosan-PRP in vivo leads to increased biological activity compared to PRP alone. We acknowledge the technical contributions of Geneviève Picard, the funding sources (CIHR, CFI, GRSTB, NSERC) and the assistance of Ortho Regenerative Technologies Inc.

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO−, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO− (25–300µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000µM stressor. Concentrations of ONOO− affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO− (threshold conc. 100µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO− (threshold conc. 100µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO− concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO− were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO− (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO−, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO− concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation. In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.

Peroxynitrite – altered platelet mitochondria—A new link between inflammation and hemostasis

Using porcine blood, we tested the hypothesis that peroxynitrite (ONOO(-)) may affect platelet-fibrin clot formation, clot retraction rate (CRR) and fibrinolysis through the inhibition of platelet energy production. It was found that ONOO(-) reduces CRR and enlarges final clot size in platelet rich plasma (PRP) (IC(50)=100μM) and in whole blood (IC(50)=200μM) dose-dependently. In a reconstituted system (washed platelets+fibrinogen), CRR was inhibited by 5-100nM ONOO(-) (IC(50)=25nM). Concentrations of ONOO(-) reducing CRR in PRP, inhibited platelet oxygen consumption, augmented lactate production and decreased total ATP contents in clots derived from PRP. In washed platelets ONOO(-) (5-20nM) produced a drop of the mitochondrial transmembrane potential (ΔΨ(m)). Blocking of mitochondrial energy production resulted in a reduction of CRR, whereas inhibition of glycolysis failed to affect CRR. ONOO(-), up to 300μM, failed to affect coagulation in platelet free plasma. Fibrinolysis of platelet-fibrin clots was enhanced by ONOO(-) (25-300μM), cytochalasin B and following the reduction of platelet energy production. Fibrinolysis of plasma clots was resistant to ONOO(-) treatment up to a concentration of 500μM. Tromboelastometry (ROTEM) measurements performed in PRP show that inhibition of platelet energy production or treatment with ONOO(-) (100-300μM) diminishes MCF, alpha angle and MCE parameters. Blockage the platelet contractile apparatus by cytochalasin B resulted in reduction of CRR and ROTEM variables (MCF, alpha angle, MCE). We conclude that physiologically relevant ONOO(-) concentrations may inhibit clot retraction, reduce clot stability and accelerate its lysis through the inhibition of platelet mitochondrial energy production.

Isolation, amino acid sequence and biological characterization of an “aspartic-49” phospholipase A2 from Bothrops (Rhinocerophis) ammodytoides venom

A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD50 of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD50 of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.

Immunoglobulin M antiplatelet autoantibodies from mice immunized with rat platelets induce thrombocytopenia and platelet function impairment

Antiplatelet monoclonal autoantibodies (mAbs) were derived from CBA mice immunized with rat platelets. Two IgM antiplatelet mAbs were further analyzed. L1C43 mAb bound a 150-160 kDa antigen, recognized activated platelets better than resting ones and impaired platelet adhesion, but not clot retraction. L1H31 mAb recognized a ±95 kDa molecule, bound similarly activated and resting platelets and did not modify platelet adhesion, but inhibited clot retraction. Both mAbs induced in vivo thrombocytopenia most likely through phagocytosis of opsonized platelets. These autoreactive antibodies can thus induce both platelet destruction and impairment of their function. They are reflective of autoantibodies found in human patients and may serve for further analysis of antiplatelet autoantibody pathogenicity and mechanisms of autoimmune disease.

Comparative screening of plant essential oils: Phenylpropanoid moiety as basic core for antiplatelet activity

Essential oils extracted from different plants ( Anthemis nobilis L., Artemisia dracunculus L., Cannabis sativa L., Cupressus sempervirens L., Cymbopogon citratus (DC.) Stapf., Curcuma longa L ., Foeniculum vulgare L., Hypericum perforatum L., Hyssopus officinalis L., Mentha spicata L., Monarda didyma L., Ocimum basilicum L ., Ocotea quixos Kosterm ., Origanum vulgare L., Pinus nigra J.F. Arnold, Pinus silvestris L., Piper crassinervium Kunth., Rosmarinus officinalis L ., Salvia officinalis L., Salvia sclarea L., Santolina chamaecyparissus L., Thymus vulgaris L., Zingiber officinaie L .) were screened in guinea pig and rat plasma in order to assess antiplatelet activity and inhibition of clot retraction. The oils were chemically analysed and a relationship between components and ability to affect hemostasis was evidenced. O. quixos, F. vulgaris, and A. dracunculus showed the highest antiplatelet activity against ADP, Arachidonic Acid and the Thromboxane A 2 agonist U46619 (IC 50, 4–132 μg ml − 1 ), and a good ability to destabilize clot retraction (IC 50, 19–180 μg ml − 1 ). For these oils a significant correlation between antiplatelet potency and phenylpropanoids content (54–86%) was evidenced thus suggesting a key role for this moiety in the prevention of clot formation. These findings provide the rationale to take in account the antiplatelet activity in the pharmacological screening of natural products containing phenylpropanoids.

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.

Regulation of clot retraction by glycoprotein IIb/IIIa antagonists

Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa induces clot retraction. Significant differences among GP IIb/IIIa antagonists were previously noted to inhibit thromboelastography in whole blood specimens. The relationship between efficacy of these agents and inhibition of clot retraction is unclear. Here, we use a plasma-free clot retraction assay to evaluate potency of GP IIb/IIIa antagonists to inhibit clot retraction and modulate platelet signaling, and to address whether these effects are realized in the clinically relevant dose range. The potencies for inhibition of clot retraction and aggregation are similar for antagonists with high affinity for resting platelets and slow off-rates, whereas lower affinity and fast off-rate antagonists are disproportionately less effective in blocking clot retraction. A positive correlation is observed between inhibition of clot retraction and inhibition of tyrosine dephosphorylation across a number of GP IIb/IIIa antagonist pharmacophores. For lower affinity and fast off-rate antagonists, the concentrations required for inhibition of clot retraction clearly exceed the clinical dose range. Site occupancy studies combined with clot retraction experiments addressed whether high affinity and slow off-rate compounds can alter clot retraction during the dosing interval. Binding studies using [ 3H] Roxifiban, a high affinity GP IIb/IIIa antagonist, indicate that occupancy of >95% of GP IIb/IIIa sites is required to inhibit clot retraction. This level of occupancy is not routinely achieved in the clinic and is not tolerated, at least for chronic therapy. These results suggest that inhibition of clot retraction is not necessary for efficacy of GP IIb/IIIa antagonists.

Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization

Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction; however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADP addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa– mediated ligand binding can be separated mechanistically from GPIIb-IIIa–mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.