Involvement of Src kinases and PLCγ2 in clot retraction

SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.

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Essential Athrombia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647881 ◽

1975 ◽

Vol 33 (02) ◽

pp. 278-285 ◽

Cited By ~ 3

Author(s):

Şeref Inceman ◽

Yücel Tangün

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Bleeding Tendency ◽

Clot Retraction ◽

Platelet Factor ◽

Prolonged Bleeding Time ◽

Normal Platelet ◽

Aggregation Response ◽

Platelet Disorder ◽

Fibrinogen Content

SummaryA constitutional platelet function disorder in a twelve-year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported.Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1–4 μM), although a small wave of primary aggregation was obtained by very large ADP concentrations (25–50 μM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Pelease of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal.The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of “essential athrombia.”

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Studies on the Haemostatic Defect in a Complicated Syndrome

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649920 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1244-1251 ◽

Cited By ~ 37

Author(s):

H Stormorken ◽

H Holmsen ◽

R Sund ◽

K S Sakariassen ◽

T Hovig ◽

...

Keyword(s):

Ex Vivo ◽

Bleeding Tendency ◽

Clot Retraction ◽

Abnormal Finding ◽

Shear Rates ◽

Perfusion Chamber ◽

Coagulant Activity ◽

5 Ht Uptake ◽

And Storage

SummaryThe Stormorken syndrome is a multifacetted syndrome including a bleeding tendency. No deviations were found in the coagulation- or fibrinolytic systems. Platelet number was low normal, and size abnormal, whereas EM findings were unremarkable. Survival time was half normal. Clot retraction was initially rapid, but clearly decreased, whereas prothrombin consumption was also initially rapid, but complete. Membrane GP’s were normal, so was AA metabolism, PI-cycle, granule storage and secretion, and c-AMP function, whereas 5-HT uptake and storage was decreased. Optical platelet aggregation was low normal with all physiological agonists. The only clearly abnormal finding was that coagulant activity was present on non stimulated platelets at the same level as kaolin-stimulated normal platelets. This indicated a platelet abnormality which should lead to a thrombogenic, not to a haemorrhagic trait. This paradox may have its origin in rheology, because when challenged with in vivo shear rates in an ex vivo perfusion chamber, platelet cohesion was abnormally low. Further studies to better delineate the membrane abnormality are underway.

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Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648803 ◽

1994 ◽

Vol 72 (01) ◽

pp. 001-015 ◽

Cited By ~ 124

Author(s):

Juan J Calvete

Keyword(s):

Structure Function ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Primary Role ◽

Clot Retraction ◽

Inhibitory Peptide ◽

Rgd Sequence ◽

Platelet Receptor ◽

Adhesive Proteins ◽

And Function

SummaryThe glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.

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Clot Retraction in von Willebrand’s Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649193 ◽

1974 ◽

Vol 31 (03) ◽

pp. 519-520

Author(s):

Leo R. Zacharski ◽

Robert Rosenstein

Keyword(s):

Clot Retraction ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease

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Effects of Prostaglandins, Derivatives of Cyclic 3':5'-AMP, Theophylline, Cholinergic Agents and Colchicine on Clot Retraction in Dilute Platelet-Rich Plasma and Gel-Separated Platelet Test Systems

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651477 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0420-0428 ◽

Cited By ~ 1

Author(s):

J. L Moake ◽

P. L Cimo ◽

K Widmer ◽

D. M Peterson ◽

J. R Gum

Keyword(s):

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Fibrin Clot ◽

Serotonin Release ◽

Dibutyryl Camp ◽

Clot Retraction ◽

Dilute Suspensions ◽

Cholinergic Agents ◽

Camp Levels ◽

Derivatives Of

SummaryIn dilute suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP), dibutyryl-cAMP (DBcAMP) and monobutyryl-cAMP inhibited platelet-mediated fibrin clot retraction in concentrations of 2–3 × 10–6M, with complete inhibition at 1–3 × 10–4M. Prostaglandin E1 (PGE1), which inhibited fibrin clot retraction in concentrations greater than 1.5–3 × 10–8M, was a more effective inhibitor than either PGE2 or PGF2α. In the presence of theophylline (10–4M), concentrations of DBcAMP, PGE1 PGE2 and PGF2α necessary to inhibit fibrin clot retraction were reduced 50-fold for DBcAMP and 2.5 to 20-fold for the prostaglandins. In dilute PRP or GSP, inhibition of fibrin clot retraction does not result from inhibition of thrombin-induced platelet aggregation. Thus, compounds which increase platelet cAMP levels result in the inhibition of platelet-mediated fibrin clot retraction, and this inhibitory effect may be mediated, at least in part, through suppression of platelet contractility. Cyclic GMP, dibutyryl-cGMP and carbamylcholine-Cl (which stimulates guanylate cyclase) did not influence fibrin clot retraction, and did not prevent inhibition of fibrin clot retraction by DBcAMP and PGE?. Colchicine, in concentrations known to disrupt platelet microtubules (2.5 × 10–6M to 2.5 x 10–3M), had little inhibitory effect on either fibrin clot retraction or platelet (3H)-serotonin release.

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Daunorubicin and Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650125 ◽

1981 ◽

Vol 45 (01) ◽

pp. 038-042 ◽

Cited By ~ 9

Author(s):

M E Pogliani ◽

R Fantasia ◽

G Lambertenghi-Deliliers ◽

E Cofrancesco

Keyword(s):

Electron Microscope ◽

Cellular Membrane ◽

Hemorrhagic Diathesis ◽

Clot Retraction ◽

Freezing And Thawing ◽

Platelet Factor ◽

High Doses ◽

Very High ◽

Platelet Functions

SummaryThe influence of Daunorubicin on some platelet functions in vitro was investigated, using different concentrations of the drug (0.01-0.02-0.04 μg/ml). Daunorubicin was shown to inhibit Collagen and Thrombin induced platelet aggregation and the intensity of inhibition depended on both drug concentration and the time of preincubation.Daunorubicin was also shown to inhibit the release reaction, the platelet prostaglandin pathway and the availability platelet factor 3; the drug at concentrations for clinical use does not damage the platelet membrane, as is the case with the freezing and thawing test, in platelet uptake of 14C-serotonin and as confirmed by the electron microscope. When very high doses (0.16 mg) of Daunorubicin are used, lysis of the platelets can be observed and this is confirmed under the electron microscope by the presence of empty platelets with fractures at the level of the cytoplasmic membrane.Finally, Daunorubicin causes irreversible inhibition of reptilase clot-retraction, even if this is less severe than with Vincristine. Working with gel-filtered platelets, it would appear that the inhibition exercised by the drug on platelet reactions is not caused through modifications in Ca++ metabolism.The authors suggest that Daunorubicin, at the dosages used clinically, induces in vitro thrombocytopathy without damaging the cellular membrane as confirmed by the electron microscope.This impairment of platelet functions could play a part in hemorrhagic diathesis observed during Daunorubicin therapy.

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Preservation of Morphological Integrity and Clot Retraction Activity of Human Platelets After Freezing

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654822 ◽

1964 ◽

Vol 11 (01) ◽

pp. 222-229 ◽

Cited By ~ 8

Author(s):

Isaac Djerassi ◽

Albert Roy ◽

Jorge Alvarado ◽

Keyword(s):

Dimethyl Sulfoxide ◽

Liquid Nitrogen ◽

Human Platelets ◽

Slow Freezing ◽

Critical Conditions ◽

Plasma Medium ◽

Clot Retraction ◽

Controlled Freezing ◽

Direct Immersion

SummaryHuman platelets frozen at −195° C (liquid nitrogen) retain their morphological integrity and ability to promote clot retraction when 5% dimethyl-sulfoxide and 5% dextrose are added to the suspending plasma medium. Slow freezing was more effective than direct immersion in the liquid nitrogen. Although similar results may be achieved with dimethylsulfoxide alone with rigidly controlled freezing rates, the addition of sugars may permit freezing under less critical conditions.Dimethylsulfoxyd und 5% Dextrose dem Plasmamilieu hinzugefügt werden. Das langsame Einfrieren ist effektiver als das direkte Eintauchen in flüssigen Stickstoff. Obschon ähnliche Resultate mit Dimethylsulfoxyd allein unter exakter Kontrolle der Einfrierungsgeschwindig-keit erreicht werden können, erlaubt die Zugabe von Dextrose ein Einfrieren unter weniger kritischen Bedingungen.

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The Influence of Serotonin on Clot Retraction and the Thrombelastogram

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656266 ◽

1958 ◽

Vol 02 (01/02) ◽

pp. 111-124 ◽

Cited By ~ 2

Author(s):

E Deutsch ◽

K Martiny

Keyword(s):

Human Plasma ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Specific Effect ◽

High Dose ◽

Clot Retraction ◽

Platelet Counts ◽

Essential Value ◽

High Doses ◽

Free Plasma

Summary1. Normal platelets are necessary for induction of normal clot retraction.2. Serotonin does not induce retraction in human platelet-free plasma-clots or enhance clot firmness as measured in the coagulogram.3. Serotonin does not improve clot retraction or firmness in plasma clots with sub-optimal platelet counts.4. Methylserotonin inhibits clot retraction of platelet-rich plasma to a certain extent in moderate doses, whereas, high doses are ineffective. BOL 148 has a similar, but less significant action. There is a possibility that these effects are specific antiserotonin-effects.5. LSD 25 was ineffective in all concentrations used.6. Largactil and reserpin inhibit retraction in high doses. There seems to be a non specific effect caused by the high dose.7. Reserpine does not release a retraction-inducing agent from the platelets, which could be detected in the centrifuged platelet-free plasma used for the incubation.8. Serotonin does not replace the retraction-cofactor of Hartert, or the dialyzable factor of Lüscher in synthetic clotting substrates.9. Serotonin is of no essential value in inducing normal retraction of human plasma clots.

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Detection of Carriers in Glanzmann's Thrombasthenia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657357 ◽

1983 ◽

Vol 49 (03) ◽

pp. 182-186

Author(s):

G T E Zonneveld ◽

E F van Leeuwen ◽

A Sturk ◽

J W ten Cate

Keyword(s):

Bleeding Time ◽

Periodic Acid ◽

Type I ◽

Clot Retraction ◽

Periodic Acid Schiff ◽

Glanzmann’S Thrombasthenia ◽

Aggregation Response ◽

Glanzmann's Thrombasthenia ◽

Sds Page ◽

Reduced State

SummaryQuantitative glycoprotein (GP) analysis of whole platelets or platelet membranes was performed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and periodic acid Schiff staining in the families of two unrelated Glanzmann’s thrombasthenia (GT) patients. Each family consisted of two symptom free parents, a symptom free daughter and a GT daughter. All symptom free members had a normal bleeding time, clot retraction and platelet aggregation response to adenosine 5’-diphosphate (ADP), collagen and adrenalin. Platelet Zw* antigen was normally expressed in these subjects. GT patiens, classified as a type I and II subject, showed reduced amounts of GP lib and of GP nia. Analysis of isolated membranes in the non-reduced state, however, showed that the amount of GP Ilia was also reduced in three of the four parents, whereas one parent (of the GT type I patient) and the two unaffected daughters had normal amounts of GP Ilia. Quantitative SDS-PAGE may therefore provide a method for the detection of asymptomatic carriers in GT type I and II.

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