Platelet-Bound Prekallikrein Promotes Pro-Urokinase-Induced Clot Lysis: A Mechanism for Targeting the Factor XII Dependent Intrinsic Pathway of Fibrinolysis

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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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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Clot retraction facilitates clot lysis

Blood ◽

10.1182/blood.v57.1.44.44 ◽

1981 ◽

Vol 57 (1) ◽

pp. 44-48 ◽

Cited By ~ 25

Author(s):

RC Carroll ◽

JM Gerrard ◽

JM Gilliam

Keyword(s):

Deep Vein Thrombosis ◽

Cytochalasin B ◽

Platelet Rich Plasma ◽

Clot Lysis ◽

Clot Retraction ◽

Similar Extent ◽

Platelet Poor Plasma ◽

Vein Thrombosis ◽

Deep Vein

Abstract Platelet facilitation of clot lysis was studied using the dilute clot lysis assay, a standardized assay for fibrinolysis shown to correlate with the development of postoperative deep vein thrombosis. Clots prepared from dilute platelet poor plasma showed prolonged clot lysis when compared with clots prepared in a similar fashion from dilute platelet rich plasma. Since in the presence of platelets clot retraction or contraction occurred, we evaluated a possible direct contribution of retraction to clot lysis. Dilute platelet poor plasma clots were compacted by centrifugation, to a similar extent as that achieved during clot retraction in dilute platelet rich plasma. These clots now lysed at a rate that approached that seen with dilute platelet rich plasma clots. Using an alternate alternate approach, dilute platelet rich plasma clots were treated with cytochalasin B to prevent clot retraction. Such clots now showed prolonged lysis similar to that seen with dilute platelet poor plasma. The prolonged lysis of cytochalasin B treated dilute platelet rich plasma clots was corrected by artificial compaction of the clots. The results suggest that clot retraction markedly facilitates clot lysis, and shows that a major role of platelets to facilitate clot lysis is the effect of these cells to cause clot retraction.

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Circulating Human Platelets Express LRP-1 and uPAR mRNAs and Synthesize the Proteins: The Complex LRP-1, uPAR, PAI-1 and uPA Play a Role in Modulating Fibrinolysis in Platelet-Rich Thrombi

Blood ◽

10.1182/blood.v120.21.1116.1116 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1116-1116

Author(s):

Olga Panes ◽

Valeria Matus ◽

César González ◽

Claudia G Sáez ◽

Jaime Pereira ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Platelet Membrane ◽

Fibrinolytic System ◽

Clot Lysis ◽

Clot Retraction ◽

Tetrameric Complex ◽

Pai 1

Abstract Abstract 1116 Platelets are intrinsic components of hemostatic and pathological clots, and are essential for clot retraction. However, their role and sequential involvement in clot stabilization and lysis are still poorly understood. Human platelets contain several components of the fibrinolytic system, including functional PAI-1, TAFI, uPA and α 2-antiplasmin. Moreover, platelets possess a rich transcriptome and synthesize several proteins, among them, PAI-1. Using a global, modified clot lysis time assay in platelet-rich plasma (CLT-PRP; Panes et al., Platelets 2012) we found that the CLT-PRP was significantly longer than that of CLT in platelet-free plasma (PFP), reflecting a down-regulation of the fibrinolytic process. However, the prolonged CLT in subjects receiving tranexamic acid was normalized earlier in PRP than in PPP, denoting some pro-fibrinolytic activity in clots formed in a platelet milieu. Aim: to study the presence, origin, association and functional role of components of the fibrinolytic system in human platelets. Also, we aim to getting insight into the dynamic balance and modulation of the fibrinolytic process by the interplay of pro- and anti-fibrinolytic platelet factors. Methods and Results: in washed, leukocyte-free human platelets we detected expression of LRP-1, uPAR, PAI-1 mRNAs, and synthesis of these proteins (metabolic radiolabeling). Neither uPA mRNA nor synthesis of uPA was evidenced. All of these proteins, including uPA were detected in membrane or cytosol fractions by western blotting (WB). LRP-1 and uPAR were present in the outer leaflet of platelet membranes, with increased uPAR labeling after platelet activation (confocal microscopy-immunofluorescence). Non-stimulated whole platelets exhibit a low basal uPA activity (specific chromogenic substrate) selectively inhibited by amiloride. uPA activity falls slightly immediately after VWF-Ristocetin (VWF-R) and TRAP stimulation, but recovers to basal levels after 15min. Biotinylated washed platelets were immunoprecipitated (IP) with α -uPAR MoAb at different times before and after activation with either TRAP or VWF-Ristocetin. Co-precipitations with LRP-1, PAI-1 and uPA were detected in WB only after platelet activation with TRAP for 5 min, denoting the formation of a tetrameric complex, likely involved in endocytosis and receptor recycling. Interestingly, 5min after TRAP stimulation, uPA was sharply reduced, disappearing at 15 min, either in membrane or cytosol fractions, suggesting degradation of the protein. Similar pattern of co-precipitations were observed when IP was done with α -LRP-1 MoAb. Co-precipitations were more prominent in purified platelet membrane than in cytosolic fractions. Conclusions: human platelets express LRP-1, uPAR and PAI-1 mRNAs, and synthesize these proteins. uPA activity is present in whole, purified, washed platelets, and the protein is likely bound to the external platelet membrane. Co-precipitation of all these fibrinolytic components presumably denotes the formation of a tetrameric complex with endocytic and recycling capacities, as demonstrated in other cell lineages. Sequential IP′s after platelet activation disclose the disappearance of uPA, but not of PAI-1, from the complex, probably explained by a degradation process. Taken together, these results suggest that platelets play a predominantly antifibrinolytic role during early stages of formation of platelet-rich clots. Disclosures: No relevant conflicts of interest to declare.

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Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Blood ◽

10.1182/blood-2010-07-290551 ◽

2011 ◽

Vol 117 (15) ◽

pp. 4134-4141 ◽

Cited By ~ 31

Author(s):

Jessica L. MacQuarrie ◽

Alan R. Stafford ◽

Jonathan W. Yau ◽

Beverly A. Leslie ◽

Trang T. Vu ◽

...

Keyword(s):

Partial Thromboplastin Time ◽

Activated Partial Thromboplastin Time ◽

Factor Xii ◽

Intrinsic Pathway ◽

High Affinity ◽

Factor Xiia ◽

Platelet Release ◽

Contact Pathway ◽

Physiologic Role

Abstract Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2μM and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentration-dependent fashion but has no effect on tissue factor–induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn2+, but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa–mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn2+ will promote this interaction.

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Human Prekallikrein (Plasminogen Proactivator): Purification, Characterization and Activation by Activated Factor XII

10.1055/s-0039-1682694 ◽

1977 ◽

Author(s):

Bonno N. Bouma ◽

John H. Griffin

Keyword(s):

Human Plasma ◽

Plasminogen Activator ◽

Clot Lysis ◽

Factor Xii ◽

Globulin Fraction ◽

Factor Xi ◽

Form Analysis ◽

Plasminogen Activator Activity ◽

Fibrin Plate ◽

Factor Xiia

In order to resolve conflicting reports about the possible identity of prekallikrein and Factor XII-dependent plasminogen proactivator (FXII-PPA), the γ-globulin fractions of prekallikrein-deficient (Fletcher trait) and of normal plasma were assayed for FXII-PPA. Based on both fibrin plate and clot lysis tests, FXII-PPA in the γ-globulin fractions of prekallikrein-deficient plasmas from 2 unrelated patients was undetectable, i.e. <1% of the FXII-PPA in the normal γ-globulin fraction. However, PPA independent of FXII was detected in both the Fletcher and the normal γ-globulin fractions at 4% of the FXII-PPA present in the normal γ-globulin fraction.Human plasma prekallikrein was purified 2,000-fold (specific clotting activity 22 units/mg) and was greater than 95% homogeneous on SDS-gels. FXII-PPA was always copurified with prekallikrein and was totally separated from Factor XI. No Factor XII-dependent or Factor XII-independent plasminogen activator activity was detected in purified Factor XI preparations at 40 units/ml. Purified prekallikrein in its precursor form gave 2 protein bands on SDS-gels at 82,000 and 78,000 MW. Upon reduction, a single 85,000 MW band was observed. Kallikrein and plasminogen activator activity were generated upon incubation with purified human Factor XIIa (28,000 MW form). Analysis of this reaction mixture on SDS-gels without reduction showed 2 bands with apparently identical MW’s as the precursor protein bands, whereas reduction showed cleavage of both protein bands.These results suggest that prekallikrein is identical to FXII-PPA in normal human plasma and that activation of this zymogen by Factor XIIa involves limited proteolytic cleavage.

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Absent Plasma Second Phase Platelet Aggregation Factor in a Patient with Hageman Trait

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654068 ◽

1970 ◽

Vol 23 (03) ◽

pp. 423-431 ◽

Cited By ~ 1

Author(s):

P Okonkwo ◽

R Post ◽

H Sise

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Second Phase ◽

Minor Bleeding ◽

Factor Xii ◽

Bleeding Tendency ◽

Clot Retraction ◽

Platelet Poor Plasma ◽

Normal Platelet ◽

Normal Individuals

SummaryA case is described of a patient with factor XII deficiency in whom the second phase of platelet aggregation was missing after addition of ADP to platelet rich plasma. There was very little aggregation on addition of epinephrine, collagen or thrombin. Clot retraction was absent, but all other platelet functions were normal and there was a very minor bleeding tendency. The defect was corrected by normal platelet poor plasma and was found to be due to a missing cofactor of platelet aggregation normally found in fraction 1–0. The defect could be distinguished from that seen in subjects ingesting aspirin as well as from the defect seen in patients with a long bleeding time, defective platelet aggregation with ADP, epinephrine and collagen at low concentrations, no correction by addition of normal platelet poor plasma, and a history of mild bleeding disorder. It is thought that this defect may exist in otherwise normal individuals and that attention in this case was called to it by the presence of the deficiency of factor XII. It could be conclusively shown that factor XII is not necessary for normal platelet aggregation.

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Haemostasis and High Altitude

10.1055/s-0039-1682425 ◽

1977 ◽

Author(s):

I. Singh ◽

I.S. Chohan

Keyword(s):

Factor Viii ◽

High Altitude ◽

Bleeding Time ◽

Clot Lysis ◽

Factor Xii ◽

Factor V ◽

Platelet Adhesiveness ◽

Clot Retraction ◽

Platelet Factor ◽

Lysis Time

On arrival at high altitude there is a tendency towards hypercoaguiation associated with an increase in platelet count, factors X and XII, and thrombo-test activity (TA) which is reflected by decrease in prothrombin time (PT), bleeding time (BT), clotting time in glass (CT-gl) and in silicone (CT-sl), and stypven time (ST). Clot retraction is impaired. This hypercoaguiation state is countered by a compensatory rise in fibrinolytic activity reflected by reduction of clot lysis time (CLT), plasma fibrinogen, and factor VIII.This hypercoaguiation state persists throughout the early fortnight after arrival at high altitude and then starts regressing. On day 3, factor V decreases, BT, and ST are further reduced, and factor VIII shows a rise. On day 7, a progressive rise occurs in factors V, VIII, X and XII, TA, platelet counts and platelet factor 3 (PF-3).CT-gl, CT-sl, PT and ST are further shortened. On day 14, haematocrit rises and of all the parameters, factors V and X and clot retraction return to normal. Throughout the fortnight, factor XII remains high, CLT is short, and platelet adhesiveness remains within normal range.After 2 years’ stay at high altitude, a regression in hypercoagulability occurs and is indicated by persistent short CLT, prolonged BT, CT-sl, PT, ST and reduced TA. Platelet adhesiveness, PF-3, factors V, VIII and XII and clot retraction are restored to normal.

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Factor XIIa Is a Kinetically Favorable Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614326 ◽

1999 ◽

Vol 82 (09) ◽

pp. 1041-1046 ◽

Cited By ~ 25

Author(s):

Karina Feddersen ◽

Rasmus Røjkjær ◽

Inger Schousboe

Keyword(s):

Competitive Inhibitor ◽

Assay System ◽

Clot Lysis ◽

Factor Xii ◽

Physiological Concentration ◽

Lysis Time ◽

Factor Xiia ◽

Specificity Constant ◽

Endpoint Assay

SummaryInitiation of the plasma contact system has been shown to play a significant role in the fibrinolysis, activating both pro-urokinase and plasminogen. The aim of the present study was to further evaluate the functional role of the factor XIIa catalyzed activation of plasminogen. Activation of plasminogen by factor XIIa followed the Michaelis-Menten rate equation. In a continuous assay system the Km was 0.27 μΜ; the kcat 0.078 min−1 and the kcat/Km 0.31 × 106 Μ−1 × min−1. In an endpoint assay system the Km was 0.58 μΜ; the kcat 0.096 min−1 and the kcat/Km 0.16 × 106 Μ−1 × min−1. The discrepancy between the kcat in the two assays is not significant. Theoretically the higher Km in the end-point assay system may be due to the presence or generation of an unidentified competitive inhibitor in this assay system. Comparing the catalytic constants of factor XIIa with those of urokinase activation of plasminogen, the specificity constant, kcat/Km, of factor XIIa activation of plasminogen was 20-fold lower. However, taking the low physiological concentration of urokinase into account, the efficiency of activated factor XII is equivalent to that of urokinase. When monitoring factor XIIa activation of plasminogen in a clot lysis assay, the clot lysis time was 2- to 4-fold as long as that accommodated by urokinase at factor XIIa concentrations equivalent with 5-20% of the zymogen concentration in plasma. The factor XIIa mediated clot lysis was prevented completely by the presence of a polyclonal antibody to factor XII.

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Clot retraction facilitates clot lysis

Blood ◽

10.1182/blood.v57.1.44.bloodjournal57144 ◽

1981 ◽

Vol 57 (1) ◽

pp. 44-48

Author(s):

RC Carroll ◽

JM Gerrard ◽

JM Gilliam

Keyword(s):

Deep Vein Thrombosis ◽

Cytochalasin B ◽

Platelet Rich Plasma ◽

Clot Lysis ◽

Clot Retraction ◽

Similar Extent ◽

Platelet Poor Plasma ◽

Vein Thrombosis ◽

Deep Vein

Platelet facilitation of clot lysis was studied using the dilute clot lysis assay, a standardized assay for fibrinolysis shown to correlate with the development of postoperative deep vein thrombosis. Clots prepared from dilute platelet poor plasma showed prolonged clot lysis when compared with clots prepared in a similar fashion from dilute platelet rich plasma. Since in the presence of platelets clot retraction or contraction occurred, we evaluated a possible direct contribution of retraction to clot lysis. Dilute platelet poor plasma clots were compacted by centrifugation, to a similar extent as that achieved during clot retraction in dilute platelet rich plasma. These clots now lysed at a rate that approached that seen with dilute platelet rich plasma clots. Using an alternate alternate approach, dilute platelet rich plasma clots were treated with cytochalasin B to prevent clot retraction. Such clots now showed prolonged lysis similar to that seen with dilute platelet poor plasma. The prolonged lysis of cytochalasin B treated dilute platelet rich plasma clots was corrected by artificial compaction of the clots. The results suggest that clot retraction markedly facilitates clot lysis, and shows that a major role of platelets to facilitate clot lysis is the effect of these cells to cause clot retraction.

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ADP, thrombin, and Bothrops atrox thrombinlike enzyme in platelet-dependent fibrin retraction

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1975.229.3.737 ◽

1975 ◽

Vol 229 (3) ◽

pp. 737-745 ◽

Cited By ~ 20

Author(s):

S Niewiarowski ◽

GJ Stewart ◽

N Nath ◽

AT Sha ◽

GE Lieberman

Keyword(s):

Platelet Rich Plasma ◽

Adenine Nucleotides ◽

Close Association ◽

Creatine Phosphate ◽

Fibrin Clot ◽

Human Platelets ◽

Clot Retraction ◽

Low Concentrations ◽

Mixture Prior ◽

Bothrops Atrox

Clots formed upon the addition of thrombin to human platelet-rich plasma (PRP) retracted readily but the clotting enzyme from Bothrops atrox venom did not cause retraction in PRP unless ADP, collagen, epinephrine, or low concentrations of thrombin (0.1 U) were added. The latter type of retraction was inhibited by apyrase and creatine phosphate kinase in the presence of creatine phosphate, but that induced with higher concentration of thrombin (2 U) was not. In a system composed of washed human platelets and purified fibrinogen, Bothrops marajoensis (BM) thrombinlike enzyme (highly purified preparations of viper venom) did not cause clot retraction. Addition of ADP to the platelet-fibrinogen mixture prior to BM enzyme resulted in stimulation of clot retraction that could be dissociated from the release of platelet constituents. Addition of low concentrations of thrombin (0.1 U/ml) caused retraction associated with a considerable release of adenine nucleotides that was inhibited by potato apyrase. Electron micrographs showed platelet-fibrin aggregates in all types of retracted clots. Nonretracted clots formed in the presence of potato apyrase contained discoidal platelets that were not in close association with fibrin. It has been postulated that platelet-dependent fibrin clot retraction induced by collagen, epinephrine, and low concentration of thrombin is mediated by ADP. High concentrations of thormbin may possibly promote clot retraction independently of ADP.

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