Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E1 and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.

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Factors Affecting the Factor X Activator Activity of Human Platelets

10.1055/s-0039-1682790 ◽

1977 ◽

Author(s):

J. Vermylen ◽

N. Semeraro

Keyword(s):

Factor Viii ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Activate Factor ◽

Factor Viii Inhibitor ◽

Factor X ◽

Factors Affecting ◽

Coagulant Activity ◽

Viii Inhibitor

Several recent studies have indicated that patients with a thrombotic tendency have enhanced platelet coagulant activity. It therefore is of importance to attempt to identify factors which modify platelet coagulant activities. Recent work in our laboratory has provided evidence that human platelets possess the capacity to directly activate factor X.Adenosine-5'-diphosphate, collagen, acetylsalicylic acid and prostaglandin E1 do not modify this activity. All endotoxins studied however clearly enhanced this activity.Furthermore, infusion of ‘activated’ prothrombin concentrates in haemophiliacs with or without factor VIII inhibitor enhanced the activity of this platelet activator of factor X. The hypothesis has been put forward that this may be the mechanism through which ‘activated’ prothrombin concentrates ‘bypass’ factor VIII of IX inhibitors. Platelet isolated from platelet-rich plasma to which the ‘activated’ prothrombin concentrate had been added at a concentration approaching the maximal concentration achieved following in vivo infusion, also showed an increase in platelet coagulant activity. Work is in progress to identify the component in ‘activated’ prothrombin concentrates which enhances platelet coagulant activity.

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Complement-Dependent and Complement-Independent Interactions of Bacterial Lipopolysaccharides and Mucopeptides with Rabbit and Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646788 ◽

1979 ◽

Vol 41 (02) ◽

pp. 392-406 ◽

Cited By ~ 12

Author(s):

N Semeraro ◽

M Colucci ◽

J Vermylen

Keyword(s):

Lipid A ◽

Platelet Rich Plasma ◽

Anaerobic Bacteria ◽

Human Platelets ◽

Rabbit Serum ◽

Classical Pathway ◽

Factor V ◽

Factor X ◽

Rabbit Platelets ◽

Induced Aggregation

SummaryThe effect of 10 commercial lipopolysaccharides (LPS) and of 5 highly purified LPS with variable but defined polysaccharide content, two LPS from anaerobic bacteria, two mucopeptides and two meningococcal polysaccharides, was studied on rabbit and human platelets. All the LPS preparations induced aggregation in rabbit heparinized platelet-rich plasma (PRP) but to differing degrees. However, a preparation consisting essentially of lipid A (from Salmonella minnesota Re 595) was one of the most active. The mucopeptides were very potent whereas the meningococcal polysaccharides had no effect. The activity was abolished by inactivation of complement. The lack of ability of LPS and mucopeptides to aggregate rabbit platelets in ethyleneglycol tetraacetic acid (EGTA) – PRP suggests that the mechanism depends on activation of the classical pathway of complement. None of the bacterial products induced aggregation of human platelets.When washed rabbit platelets are mixed with complement-depleted rabbit serum and calcium chloride, generation of thrombin occurs. Washed platelets contribute to thrombin generation by providing factor V, a factor X activating activity, and possibly phospholipid (Brit. J. Haemat. 36: 107, 1977). All the LPS preparations but not the mucopeptides or meningococcal polysaccharides enhanced the rate of thrombin formation by enhancing the factor X activating activity of rabbit or human platelets.It is concluded that LPS affect rabbit platelets both by complement-dependent and complement-independent mechanisms, but human platelets only by the complement- independent pathway. Mucopeptides react with platelets only by the complement-dependent way and have no effect on human platelets.

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Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642389 ◽

1994 ◽

Vol 71 (01) ◽

pp. 091-094 ◽

Cited By ~ 15

Author(s):

M Cattaneo ◽

B Akkawat ◽

R L Kinlough-Rathbone ◽

M A Packham ◽

C Cimminiello ◽

...

Keyword(s):

Cardiovascular Disease ◽

Platelet Aggregation ◽

Prostaglandin E1 ◽

Human Platelet ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Before And After ◽

Normal Human ◽

Platelet Aggregates ◽

Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

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The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645696 ◽

1990 ◽

Vol 63 (01) ◽

pp. 112-121 ◽

Cited By ~ 18

Author(s):

David N Bell ◽

Samira Spain ◽

Harry L Goldsmith

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Mean Transit Time ◽

White Blood Cells ◽

Aggregate Size ◽

Human Platelets ◽

Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

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Lack of Stability of Aggregates after Thrombin-Induced Reaggregation of Thrombin-Degranulated Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648469 ◽

1992 ◽

Vol 67 (04) ◽

pp. 453-457 ◽

Cited By ~ 8

Author(s):

Raelene L Kinlough-Rathbone ◽

Marian A Packham ◽

Dennis W Perry ◽

J Fraser Mustard ◽

Marco Cattaneo

Keyword(s):

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Aggregate Stability ◽

Relative Importance ◽

Platelet Aggregates ◽

The Stability ◽

Von Willebrand ◽

Induced Aggregation ◽

Willebrand Factor

SummaryThe stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2C1 was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 μM) and chymotrypsin(10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymo-trypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.Electron microscope gold immunocytochemistry using antihuman fibrinogen IgG, anti-von Willebrand factor and anti-fibronectin showed a) that fibrinogen in the vacuoles of degranulated platelets was visible at focal points of platelet contact in the aggregates, but that large areas of platelet contact had no fibrinogen detectable between them; and b) in comparison to fibrinogen, little fibronectin or von Willebrand factor (vWf) was detectable in the platelets.Since the linkages between thrombin-degranulated platelets reaggregated by thrombin can be disrupted either by raising cAMP (thus making glycoprotein IIb/IIIa unavailable) or by proteolysis, these linkages are less stable than those formed between normal platelets. It might therefore be expected that platelets that take part in thrombus formation and then recirculate are likely to form less stable thrombi than platelets that have not released their granule contents.

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Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648052 ◽

1976 ◽

Vol 36 (02) ◽

pp. 376-387 ◽

Cited By ~ 3

Author(s):

Teruhiko Umetsu ◽

Kazuko Sanai ◽

Tadakatsu Kato

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Human Platelets ◽

Rabbit Platelet ◽

Rabbit Platelets ◽

Β Blocker ◽

Platelet Aggregates ◽

Induced Aggregation ◽

Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

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Preclinical studies of RUC-4, a novel platelet αIIbβ3 antagonist, in non-human primates and with human platelets

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.382 ◽

2019 ◽

Vol 3 (2-3) ◽

pp. 65-74 ◽

Cited By ~ 4

Author(s):

Spandana Vootukuri ◽

Jihong Li ◽

Mark Nedelman ◽

Craig Thomas ◽

Jiang-Kang Jiang ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Initial Slope ◽

Receptor Blockade ◽

St Segment Elevation ◽

St Segment ◽

Aggregation Inhibition ◽

Induced Aggregation ◽

Dose Dependent

AbstractIntroduction:We are developing the novel αIIbβ3 antagonist, RUC-4, for subcutaneously (SC)-administered first-point-of-medical-contact treatment for ST segment elevation myocardial infarction (STEMI).Methods:We studied the (1) pharmacokinetics (PK) of RUC-4 at 1.0, 1.93, and 3.86 mg/kg intravenous (IV), intramuscular (IM), and SC in non-human primates (NHPs); (2) impact of aspirin on RUC-4 IC50in human platelet-rich plasma (PRP); (3) effect of different anticoagulants on the RUC-4 IC50in human PRP; and (4) relationship between αIIbβ3 receptor blockade by RUC-4 and inhibition of ADP-induced platelet aggregation.Results:(1) All doses of RUC-4 were well tolerated, but animals demonstrated variable temporary bruising. IM and SC RUC-4 reached dose-dependent peak levels within 5–15 minutes, with T1/2s between 0.28 and 0.56 hours. Platelet aggregation studies in NHPs receiving IM RUC-4 demonstrated >80% inhibition of the initial slope of ADP-induced aggregation with all three doses 30 minutes post-dosing, with subsequent dose-dependent loss of inhibition over 4–5 hours. (2) The RUC-4 IC50for ADP-induced platelet aggregation was unaffected by aspirin treatment (40±9 nM vs 37±5 nM;p= 0.39). (3) The RUC-4 IC50was significantly higher in PRP prepared from D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK)-anticoagulated blood compared to citrate-anticoagulated blood using either thrombin receptor activating peptide (TRAP) (122±17 vs 66±25 nM;p= 0.05;n= 4) or ADP (102±22 vs 54±13;p<0.001;n= 5). (4) There was a close correspondence between receptor blockade and inhibition of ADP-induced platelet aggregation, with aggregation inhibition beginning with ~40% receptor blockade and becoming nearly complete at >80% receptor blockade.Discussion:Based on these results and others, RUC-4 has now progressed to formal preclinical toxicology studies.

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Effects of a Polyoxyethylene Detergent on Platelet Function

10.1055/s-0039-1682739 ◽

1977 ◽

Author(s):

P.G. Barton

Keyword(s):

Lactate Dehydrogenase ◽

Platelet Rich Plasma ◽

Secondary Phase ◽

Human Platelets ◽

Brij 58 ◽

Low Concentrations ◽

High Concentrations ◽

Glass Surfaces ◽

Induced Aggregation ◽

Membrane Destabilization

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet rich plasma but had no effect on primary aggregation.Thrombin-induced aggregation of washed human platelets suspended in Tyrode’s buffer was inhibited after incubation of cells with 4.5 × 10-6M detergent. Development of prothrombin-converting activity and efflux of [14C]-serotonin, 45Ca2+ ions and labile endoperoxides were abolished concomitantly. Aggregation of washed platelets by collagen or sodium arachidonate and the attachment of cells to clean glass surfaces were also inhibited by the same concentration of Brij 58 that inhibited thrombin aggregation. This concentration of Brij 58 did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 10-4 M, lysed the cells liberating all of their serotonin, Ca2+ and lactate dehydrogenase. These results suggest that low concentrations of Brij 58 stabilize a membrane conformation against the action of platelet stimulatory agents while high concentrations produce membrane destabilization and cell lysis. The presence of albumin (BSA) in the suspending fluid increased by tenfold the concentrations of detergent required to “elicit these effects and this could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]-acetylated Brij 58.

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Effects of gas bubbling and other forms of convection on platelets in vitro

Journal of Applied Physiology ◽

10.1152/jappl.1989.67.3.1250 ◽

1989 ◽

Vol 67 (3) ◽

pp. 1250-1255 ◽

Cited By ~ 3

Author(s):

D. M. Pickles ◽

D. Ogston ◽

A. G. MacDonald

Keyword(s):

Shear Stress ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Air Plasma ◽

Physiological Level ◽

Gentle Agitation ◽

Rocking Motion ◽

Experimental Treatments ◽

Induced Aggregation

Citrated platelet-rich human plasma was subjected to one of three experimental treatments at 37 degrees C for 15 min: stirring, bubbling (with stirring), and gentle agitation achieved by a rocking motion. The last two were “equiconvective” as judged by equilibration rates with CO2 and O2 but presumably differed in the shear stress they imposed on the cells. Stirring platelets in normal air or 5% CO2-air caused no significant aggregation. Bubbling air through platelet-rich plasma increased its pH and marked aggregation occurred. Bubbling CO2-air caused the platelet-rich plasma pH to attain its physiological level of 7.4 with less aggregation. In both cases, subsequent ADP-induced aggregation was diminished. Rocking (without stirring) in the presence of CO2-air caused negligible aggregation in platelets and an enhanced response to ADP. Because of the marked difference between the two equiconvective treatments, bubbling and rocking, the main factor in activating the human platelets is suggested to be shear stress (potentiated by high pH), with perhaps a lesser contribution from the air-plasma interface.

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Inhibition of PF4 and ßTG Release from Platelets by Piracetam

10.1055/s-0039-1687600 ◽

1979 ◽

Author(s):

R.L. Henry ◽

R.M. Nalbandian ◽

G.E. Herman ◽

T. Ho

Keyword(s):

Platelet Aggregation ◽

Normal Individual ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Complete Inhibition ◽

Clot Retraction ◽

Platelet Factor ◽

Storage Pool ◽

No Inhibition ◽

No Release

Platelet factor four (PF4) and beta-thromboglobulin (βTG) were released from human platelets alpha granules by ADP and epinephrine and measured by radioimmunoassay. Both release materials are antiheparins but PF4 is reported to be more potent. However, PF4 is released at about 1/4 the level of βTG in nanog rams/ml. Total release occurred with 5 ugm/rnl ADP in platelet-rich-plasma adjusted to 200,000 platelets/mm3 and with 1.25 × 10-5M epinephrine. No further release was found by freeze-thawing procedures. In one case, no release occurred although full aggregation proceeded normally with both mediators. Only minimal amounts were recorded after freeze-thawing indicating a storage pool deficiency of PF4 and βTG in an apparantly normal individual. Complete inhibition of PF4 and βTG release was obtained concurrently with elimination of the 2nd epinephrine wave by 6.4 × 10-4 M Piracetam. In contrast to aspirin, no inhibition of ADP, Collagen, or Ristocetin aggregation or release occurred with Piracetam. In previous work it was determined that Piracetam even at 6.4 × 10-3 M did not modify thrombin, prothrombin, or activated partial thromboplastin times. In addition, clot retraction was not modified in concentrations of Piracetam as high as 1.28 × 10-2 M known to eliminate the 2nd wave of platelet aggregation by epinephrine.

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