Bleeding risks associated with inheritance of the Quebec platelet disorder

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Heather McKay ◽  
Francine Derome ◽  
M. Anwar Haq ◽  
Susan Whittaker ◽  
Emmy Arnold ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Autosomal Dominant ◽  
Data Entry ◽  
Lifestyle Changes ◽  
Excessive Bleeding ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
Fibrinolytic Inhibitors ◽  
Deep Cuts

Abstract Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with increased urokinase-type plasminogen activator in platelets and α-granule protein degradation. To determine bleeding risks and common manifestations of QPD, a history questionnaire was developed and administered to 127 relatives in a family with QPD. Data entry was done blinded to affected and unaffected status, determined by assays for platelet urokinase-type plasminogen activator (u-PA) and fibrinogen degradation. Odds ratios (ORs), with 95% confidence intervals (CIs), were determined for items queried. Summative bleeding scores for each individual were calculated using items with OR more than 1. Mean ages (34 years; range, 1-89 years) were similar for affected (n = 23) and unaffected (n = 104) family members. Affected individuals had higher mean bleeding scores (P < .0001) and a much higher likelihood (OR > 20) of having bleeding that led to lifestyle changes, bruises that spread lower or as large or larger than an orange or both, joint bleeds, bleeding longer than 24 hours after dental extractions or deep cuts, and received or been recommended other treatments (fibrinolytic inhibitors) for bleeding. Individuals with QPD and exposure(s) to hemostatic challenges had experienced excessive bleeding only when fibrinolytic inhibitors had not been used. These data illustrate that QPD is associated with increased risks of bleeding that can be modified by fibrinolytic inhibitors.

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder

2003 ◽  
Vol 90 (08) ◽  
pp. 293-298 ◽  
Author(s):  
Prameet Sheth ◽  
Walter Kahr ◽  
Anwar Haq ◽  
Dragoslava Veljkovic ◽  
Georges Rivard ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Small Proportion ◽  
Bleeding Disorder ◽  
Phenotypic Changes ◽  
Granule Protein ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
Granule Proteins

SummaryThe Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet α–granule proteins. The increased u-PA and proteolyzed plasmino-gen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD α-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-α2-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal α-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further α-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to α-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

Antithrombotic thrombocytes: ectopic expression of urokinase-type plasminogen activator in platelets

Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 926-933 ◽  
Author(s):  
Dubravka Kufrin ◽  
Don E. Eslin ◽  
Khalil Bdeir ◽  
Juan-Carlos Murciano ◽  
Alice Kuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Ectopic Expression ◽  
Inherited Disease ◽  
Platelet Factor ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
New Strategy ◽  
Fibrinolytic Proteins

Abstract Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Walter H. A. Kahr ◽  
Shilun Zheng ◽  
Prameet M. Sheth ◽  
Menaka Pai ◽  
Alison Cowie ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Plasminogen Activator ◽  
Messenger Rna ◽  
Secretory Proteins ◽  
Plasminogen Activator Inhibitor 1 ◽  
Granule Protein ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
Pai 1

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and α-granule protein degradation. The degradation of α-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P &lt; .00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P &lt; .0008) and 100-fold greater u-PA–PAI-1 complexes (P &lt; .0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many α-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of α-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

Urokinase-Type Plasminogen Activator Production by Cultured Megakaryocytes and Blood Outgrowth Endothelial Cells in the Quebec Platelet Disorder.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1008-1008 ◽  
Author(s):  
D. Kika Veljkovic ◽  
Ping Wang ◽  
Maria Diamandis ◽  
Georges E. Rivard ◽  
Catherine P.M. Hayward
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Peripheral Blood ◽  
Culture Media ◽  
Platelet Factor ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Platelet Disorder ◽  
And Control ◽  
Pai 1

Abstract The Quebec Platelet Disorder (QPD) is an unusual α-granule protein disorder, associated with increased expression and storage of urokinase-type plasminogen activator (u-PA) in platelets and platelet-dependent, accelerated clot lysis. The increased u-PA in QPD platelets, and normal to increased levels in QPD plasma, led us to investigate the production of u-PA by cultured QPD megakaryocytes (MKs) and by QPD blood outgrowth endothelial cells (BOECs). Our objectives were to 1) characterize production of u-PA and other platelet proteins by normal and QPD MKs at different stages of differentiation, and 2) determine if the abnormal production of u-PA in the QPD extended to endothelial cells. Thrombopoietin-stimulated MKs were obtained by culturing CD34+ progenitor cells isolated from peripheral blood. BOECs were obtained from fibronectin-adherent peripheral blood mononuclear cells. Cell lysates and culture media were harvested from MKs on days 7 and 13, and from BOECs on day 11. Proteins were quantified by ELISA. MK differentiation was evaluated by flow cytometry for αIIbβ3 expression. For BOECs, endothelial lineage differentiation was assessed by immunostaining for acetylated low-density lipoprotein uptake and Ulex europaeus agglutinin binding. QPD and normal control BOECs were indistinguishable in morphology and differentiation during culture, and their content of u-PA was low (ng u-PA/mg cellular protein, mean ± SD for n = 9: QPD 3.5 ± 1.1, control 3.1 ± 1.5, p = 0.54), without detectable u-PA-PAI-1 (plasminogen activator inhibitor 1) complexes. QPD BOECs also produced normal amounts of the control secretory proteins PAI-1, multimerin 1 and von Willebrand factor (VWF). The morphology, viability, proliferation and differentiation of QPD and control MKs grown in culture were indistinguishable. By 7 days of MK culture, QPD and control cells had secreted similar, low quantities of u-PA into the culture media (ng u-PA/106cells, mean ± SD for n = 4: QPD 0.3 ± 0.1, control 0.4 ± 0.1; p = 0.18). However, by day 13 of MK culture (when most cells expressed αIIbβ3), QPD cultures contained significantly more u-PA in their culture media (ng u-PA/106 cells: QPD 20.5 ± 9.8, control 0.2 ± 0.1, p &lt; 0.05 compared to controls and to day 7 QPD samples). The increased production of u-PA in late stage QPD MK cultures was associated with increased intracellular u-PA (ng u-PA/106 cells: QPD 9.6 ± 6.3, control 0.03 ± 0.02, p &lt; 0.05) and increases in intracellular and secreted u-PA-PAI-1 complexes (p &lt; 0.05). In contrast, the levels of PAI-1, VWF, thrombospondin-1 and platelet factor 4 were normal in day 13 QPD MK cultures. Taken together, these data indicate that low levels of u-PA are normally produced by MKs and BOECs isolated from peripheral blood progenitors. The increased u-PA in differentiating QPD MKs and peripheral blood platelets, and normal levels of u-PA in QPD BOECs and early MK progenitors, indicate that the molecular defect in this disorder induces a cell-type restricted defect in u-PA regulation that is manifested during the later stages of MK differentiation. The molecular nature of this congenital defect in u-PA regulation is under investigation.

Urokinase type plasminogen activator and inhibitor type-1 plasma levels in colorectal cancer

2001 ◽  
Vol 120 (5) ◽  
pp. A599-A600 ◽  
Author(s):  
L HERSZENYI ◽  
F FARINATI ◽  
G ISTVAN ◽  
M PAOLI ◽  
G ROVERONI ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Inhibitor Type

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 247-250 ◽  
Author(s):  
H R Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rate Constants ◽  
Plasminogen Activators ◽  
Second Order ◽  
Tissue Type ◽  
Single Chain ◽  
Chain Molecules ◽  
Order Rate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

Physical Exercise Induces Enhancement of Urokinase-Type Plasminogen Activator (u-PA) Levels in Plasma

1991 ◽  
Vol 65 (01) ◽  
pp. 082-086 ◽  
Author(s):  
G Dooijewaard ◽  
A de Boer ◽  
P N C Turion ◽  
A F Cohen ◽  
D D Breimer ◽  
...  
Keyword(s):  
Physical Exercise ◽  
Plasminogen Activator ◽  
Maximal Exercise ◽  
Normal Values ◽  
Bicycle Ergometer ◽  
Tissue Type ◽  
Healthy Male ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Fibrinolytic Potential

SummaryThe enhancement of the blood fibrinolytic potential by physical exercise is generally attributed to the release of tissue-type plasminogen activator (t-PA) from the vessel wall. In this study we have investigated the possible contribution of urokinase-type plasminogen activator (u-PA).Six healthy male volunteers (age 21–25 years) were screened for their ability to perform maximal exercise for their age-group for 12 min on a bicycle ergometer. Subsequently, on one occasion they were required to remain supine for 2 h (from 8.30 a. m. onwards) and on another they performed maximal exercise (from 9.00 a.m. onwards). During exercise an increase in u-PA antigen and plasmin-activatable pro-urokinase (proUK) activity, concurrent with t-PA antigen and euglobulin t-PA activity, was observed in all six volunteers, while at rest these parameters remained unaffected. Mean u-PA- and t-PA antigen increased, respectively, from 4.2 ± 1.0 ng/ml and 5.8 ± 2.1 ng/ml before exercise to 9.8 ± 3.0 ng/ml and 18.3 ± 3.8 ng/ml (peak). Mean plasminactivatable proUK activity and t-PA activity increased, respectively, from 2.1 ± 0.4 ng/ml and 0.3 ± 0.2 ng/ml before exercise to 4.3 ± 1.7 ng/ml and 7.2 ± 4.0 ng/ml (peak). The increases were statistically significant throughout (paired t-test, pre vs post, antigen P <0.005 and activity P <0.02). After cessation of exercise u-PA and t-PA declined concurrently to normal values with a 50"/" decay in about 5 min. In conclusion, we found that both u-PA antigen and plasmin-activatable proUK activity are, concurrently with t-PA, enhanced upon exercise and, therefore, we consider that u-PA also contributes to – and co-operates in – the enhancement of the blood fibrinolytic potential and activity under these conditions.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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