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PLENARY PAPER
From the Departments of Pathology and Molecular
Medicine, and Medicine, McMaster University, Hamilton; the Hamilton
Regional Laboratory Medicine Program, Ontario; and the Department of
Hematology/Oncology, Hôpital St Justine, Montreal, Quebec,
Canada.
The Quebec platelet disorder (QPD) is an autosomal dominant
platelet disorder associated with delayed bleeding and Congenital platelet disorders are usually
associated with defective primary hemostasis.1-3 The
Quebec platelet disorder (QPD) is an autosomal dominant platelet
disorder that has unusual clinical features: it is associated with
moderate to severe delayed bleeding, that typically begins 12 to 24 hours after surgery or trauma, and its hemorrhagic manifestations can
be controlled with fibrinolytic inhibitors but not with platelet
transfusions.1,4-6 This disorder was initially designated
as factor V Quebec because of the abnormalities found in platelet
factor V of these patients.7 Two families from Quebec have
been identified with this condition, which is now known to be
associated with other platelet abnormalities that include reduced to
low-normal platelet counts, proteolytic degradation of soluble and
membrane proteins stored in platelet The cause of the QPD has been uncertain. Affected patients of
both families share a characteristic pattern of platelet Patients
Materials
Sample preparation Double-centrifuged plasma (from blood collected in 9:1 vol/vol 3.2% buffered sodium citrate), washed platelet lysates, and platelet releasates were collected from patients and controls as previously described,4,5 with the following modifications. Resting platelets were prepared using anticoagulant and wash buffers supplemented with 2 µM PGE1 and 1 mM theophylline (final concentrations). Cell counts confirmed the washed platelets contained minimal leukocyte contamination. Washed platelets were solubilized (1 × 109 platelets/mL final)5 using buffer containing 0.5% Triton X-100 and multiple protease inhibitors (0.3 µM aprotinin, 2.8 µM E-64, 10 mM EDTA, 1 µM leupeptin, 5 mM N-ethyl-maleimide, 4 mM AEBSF, 1 µM pepstatin, 100 µM 1,10-phenanthrolene monohydrate, and 100 µg/mL soybean trypsin inhibitor [STI]). For some studies, platelet lysates were prepared without the serine protease inhibitors AEBSF, aprotinin, STI, and leupeptin. Platelet releasates4,5 were prepared from washed platelets, resuspended in albumin-free Tyrode buffer (pH 7.4 with 2 mM Ca++, 1 mM Mg++, and 5 mM HEPES; 1 × 109 platelets/mL) and activated (20 minutes, 37°C) using 50 µM ADP or 2 µM calcium ionophore A23187 (samples centrifuged 2000g for 10 minutes, followed by 14 000g for 15 minutes before freezing). All samples were frozen and stored at 70°C until analyzed. K562 cells stimulated
with 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 3 nM final;
Sigma-Aldrich Canada) were used as a source of PAI-1 protein and u-PA
messenger RNA (mRNA).12
Protein and protease analyses Zymography was performed using 3% agarose substrate gels (SeaPlaque agarose; BioWhittaker Molecular Applications, Rockland, MD; in phosphate-buffered saline [PBS], pH 7.4) containing plasminogen-free fibrin or casein (1% wt/vol; Carnation Instant Skim Milk Powder, Nestle Canada, Toronto, ON), with or without added plasminogen (5 µg/mL final), similar to methods previously described.13,14 Protease activities were tested by spotting samples directly onto substrate gels, or after proteins were separated by nonreduced SDS-PAGE and renatured with 2% Triton X-100 in PBS, pH 7.4, for 1 hour. Casein gels with or without 1 mM amiloride were used for some determinations. Substrate gels were incubated with samples (37°C, 18 hours) and were photographed wet. Some samples were preincubated with protease inhibitors (same final concentrations as lysates; 20 minutes on ice) or recombinant PAI-1 (0-4000 ng/mL final after 1:1 dilution in a releasate pool, prepared from 5 QPD ionophore releasates; 1 hour, 22°C) before testing their proteolytic activity. Others were tested after immunodepletion with rabbit anti-human u-PA or control normal rabbit immunoglobulin G (IgG) bound to protein A Sepharose, similar to methods described.15Plasma samples were assayed at 1:5 to 1:100 dilutions in the u-PA ELISA. Platelet lysates were tested at 1:2.5 and larger dilutions. Data for stored, washed, and pelleted platelet lysates were pooled because they contained similar amounts of u-PA at the dilutions tested. Data for new and stored lysates were analyzed separately because the stored samples were prepared with different protease inhibitors.4,5 All samples were tested undiluted in the t-PA ELISA, which was modified to include a lower concentration (2 ng/mL) standard. Some normal samples contained less u-PA or t-PA than the lowest standard of the AD ELISA when tested at recommended and lower dilutions. These amounts were reported as "less than" values when ranges for controls were determined, and they were rounded up to the nearest measurable value to calculate means and standard deviations for controls. Active PAI-1 in platelet ionophore releasate and lysate (without added serine protease inhibitors) was assessed by measuring u-PA-PAI-1 complex generation, similar to methods previously described.16 Briefly, pooled samples of releasate and lysate, prepared from 5 control and 5 QPD donors, respectively, were incubated (30 minutes, 22°C) with or without added recombinant tcu-PA (200 ng/mL final in 20 µL sample) before measuring u-PA-PAI-1 complexes by ELISA (values expressed as an average of duplicate determinations). For studies of Analyses of platelet mRNA Total RNA was extracted from platelets and from K562 cells, as previously described.17 Complementary DNA (cDNA) synthesis was carried out on 1 µg total RNA (20 µL final volume) using oligo dT as a primer and Thermoscript (Life Technologies, Burlington, ON, Canada) reverse transcriptase (RT), as recommended by the manufacturer. Polymerase chain reaction (PCR) was performed on 2 µL cDNA reaction in a final volume of 50 µL using Platinum Taq DNA polymerase (Life Technologies). Primers (synthesized by the Central Facility, McMaster University), expected products sizes, and cycle sequences for u-PA and -actin reverse transcription--PCR were u-PA
forward, 5'-GGAATGGTCACTTTTACCG-3', u-PA reverse,
5'-CTGCCCTGAAGTCGTTAG-3', expected product 1.55 kb, 94°C at 30 seconds, 50°C at 30 seconds, 72°C at 2 minutes for 30 cycles;
-actin forward, 5'-CCTCGCCTTTGCCGATCC-3', -actin reverse,
5'-GGATCTTCATGAGGTAGTCAGTC-3', expected product 620 bp, 94°C at 30 seconds, 55°C at 30 seconds, 72°C at 1 minute for 25 cycles.
Products were analyzed on 1% agarose gels and visualized with ethidium bromide.
Because platelet fibrinogen was degraded in patients with the QPD,
their platelet releasates and lysates were screened for proteolytic
activity using fibrin substrate gels (Figure
1). Fibrinolytic activity was evident in
all QPD platelet releasates tested, but it was not detected in the same
amounts of control releasates (Figure 1A; data representative of 5 patients and 12 controls). Fibrinolytic activity in QPD platelet
releasates was inhibited by the serine protease inhibitor AEBSF (Figure
1A), but it was not blocked by EDTA, leupeptin, the cysteine protease
inhibitor E64, the aspartic protease inhibitor pepstatin, or the
metalloproteinase inhibitor phenanthrolene (not shown). Similar
fibrinolytic serine protease activity was present in lysates of QPD
resting platelets, whereas it was undetectable in the same volume of
control sample (Figure 1A). Fibrinolytic activity released by QPD
platelets was not blocked in 1:2 mixtures with normal platelet
releasate or lysate (Figure 1B), suggesting the defect was not due to
an inhibitor deficiency.
Zymograms indicated there were secretable 50-kd (major band) and 100-kd
(minor band) (Mr nonreduced) fibrinolytic enzymes in QPD
platelets that were not detectable in similar amounts of normal
platelets (Figure 2A-B shows data
representative of 5 patients and 12 controls). The activities of these
fibrinolytic enzymes were destroyed by reduction (not shown).
Comparisons of their activities on fibrin substrate gels, with and
without added plasminogen (Figure 2B), indicated the 50- and 100-kd QPD
platelet proteases had properties of plasminogen activators. QPD
platelets also contained and secreted a 33-kd plasminogen activator
that was not detected in the normal samples (Figure 2B).
ELISA and Western blots were used to determine whether the plasminogen
activators in QPD platelets were either t-PA or u-PA. Although QPD and
control plasmas contained similar amounts of t-PA, neither QPD nor
control platelets contained detectable t-PA (Table
1). Both the OS and AD u-PA ELISA
indicated there was more than 100-fold more u-PA in QPD platelets than
in normal platelets (Table 1). Furthermore, comparisons of platelet
u-PA levels in unaffected family members and family members with the
QPD indicated that only the affected patients had increased platelet
u-PA levels (Table 1; data for stored platelet samples). The OS u-PA
ELISA detected approximately 4-fold more u-PA in QPD platelets than the
AD u-PA ELISA (Table 1), suggesting these assays differed in their
ability to detect some forms of u-PA. The amounts of u-PA in normal
plasma, measured by both OS and AD u-PA ELISA (Table 1), were similar
to previously reported values.18-21 Each ELISA indicated
patients with the QPD had larger increases in u-PA in their platelets
than their plasmas because many patients had normal plasma u-PA levels
(Table 1). u-PA ELISA confirmed QPD platelets released significant
quantities of u-PA with secretagogue stimulation because their ADP
releasates contained approximately 9% of their platelet u-PA, and
their ionophore releasates contained approximately 48% of their
platelet u-PA (averaged data, AD ELISA; n = 3 patients evaluated).
Western blots (probed with monoclonal and polyclonal u-PA antibodies)
confirmed that QPD platelets and platelet releasates contained
abnormally large amounts of u-PA (Figure
3A shows data representative of 5 patients). Western blots of stored platelet lysates, from additional
affected (n = 9) and unaffected (n = 5) members of both QPD
families, confirmed this abnormality was present only in affected
patients (not shown).
Western blots were used to determine whether the u-PA in QPD platelets comigrated, nonreduced and reduced, with purified scu-PA, tcu-PA, or LMW u-PA (Figure 3). There was considerable heterogeneity in the forms of u-PA found in QPD platelet lysates, and their releasates contained identical forms (Figure 3). On nonreduced gels (Figure 3A; Figure 3B, left panel), the most abundant form of u-PA in QPD platelets comigrated with scu-PA and tcu-PA, whereas only a small proportion comigrated with LMW u-PA (Figure 3B). After reduction (Figure 3B, right panel), the most abundant form of u-PA in QPD platelets had the mobility of tcu-PA, indicating most u-PA in QPD platelets had been activated. Some of the less abundant forms of u-PA in QPD platelets were proteolyzed and did not comigrate with scu-PA, tcu-PA, or LMW u-PA (Figure 3A-B and longer exposures, not shown). A small proportion of their total u-PA was larger than scu-PA and tcu-PA and resembled high-molecular weight complexes generated by incubating exogenous scu-PA (Figure 3A, right panel) or tcu-PA (not shown) with normal platelet releasate proteins. Zymograms indicated none of the QPD platelet plasminogen activators
comigrated with t-PA or plasmin, and they confirmed the 50- and 33-kd
plasminogen activators in QPD platelets comigrated with tcu-PA and LMW
u-PA, respectively (Figure 4A). The
activities of the 100-, 50-, and 33-kd QPD platelet plasminogen
activators were blocked by 1 mM amiloride, which inhibited tcu-PA but
not t-PA activity, as previously reported22 (Figure 4B).
All the plasminogen activators in QPD releasates were neutralized when recombinant PAI-1 was added to final concentrations of 3000 ng/mL or
more (Figure 4C), which was more than the concentration of PAI-1 in
normal and QPD platelet lysates (Table 1). Furthermore, antibodies to
u-PA selectively removed all detectable plasminogen activators (Figure
4D) and fibrinolytic proteases (not shown) from QPD releasates. These
observations indicated that the fibrinolytic, plasminogen-activating
proteases detected in QPD platelets were different forms of the enzyme
u-PA.
RT-PCR analyses were performed to determine whether the u-PA
abnormalities in the QPD platelets were associated with increased u-PA
mRNA levels in platelets. Although platelets from patients and controls
contained similar amounts of
Unregulated u-PA activity in QPD platelets was further
investigated by measuring platelet PAI-1 antigen and u-PA-PAI-1
complexes using ELISA. QPD platelets contained approximately 2-fold
more PAI-1 antigen and more than 100-fold more u-PA-PAI-1 complexes than normal platelets (Table 1). Western blots confirmed some of the
PAI-1 in QPD platelets had formed complexes with u-PA, though the
proportions of complexed PAI-1 varied slightly between patients (Figure
6A; Pt 3 indicates the patient with the
highest concentrations of platelet u-PA-PAI-1 complexes by ELISA).
Increased u-PA-PAI-1 complexes were also detected in QPD platelet
releasates using ELISA (Figure 6B), but they were difficult to detect
by Western blotting (Figure 6A and analyses of larger sample volumes, not shown). The high-molecular weight PAI-1 complexes stored in QPD
platelets expressed epitopes recognized by u-PA antibodies (Figure 6A,
lane *), and they comigrated with PAI-1 complexes generated in vitro by
adding tcu-PA to normal platelet releasate (Figure 6A, right panel).
All the QPD platelets tested contained proteolyzed forms of PAI-1
(Figure 6A, arrow) that were not evident in normal platelets, but only
traces of similar proteolyzed forms were detected in control releasates
incubated with tcu-PA (Figure 6A and longer exposures, not shown).
Assays of active PAI-1 indicated that although pooled QPD platelet lysates and releasates contained abnormally large amounts of u-PA-PAI-1 complexes before exogenous u-PA was added, they were unable to generate additional complexes with exogenous u-PA (Figure 6B). Furthermore, the amounts of u-PA-PAI-1 complexes generated when u-PA was added to pooled normal releasates and lysates were similar to the amounts contained in pooled QPD releasates and lysates (Figure 6B). These data indicated the active forms of PAI-1 had been depleted in QPD platelets, likely because they had formed complexes with u-PA in vivo. Western blots were used to determine whether the changes in u-PA in the
QPD were associated with plasminogen proteolysis. QPD plasmas contained
forms and amounts of plasminogen that were indistinguishable from
normal controls (not shown). Although the plasminogen in normal, washed
platelets comigrated with purified Glu-plasminogen, in QPD platelets
much of the plasminogen was proteolyzed (Figure
7), and there was a form that comigrated
with plasmin on reduced (Figure 7) and nonreduced (not shown) gels. When normal platelet releasate was incubated with exogenous tcu-PA, there was loss of detectable intact plasminogen; however, the extent of
plasminogen proteolysis was not as complete as in QPD platelets and the
tcu-PA digests of purified plasminogen (Figure 7).
Next, we investigated whether exogenous tcu-PA (in concentrations
similar to the increased u-PA in QPD platelets) could trigger the
proteolysis of other stored platelet proteins to forms that comigrated
with degraded proteins in QPD platelets (Figures
8, 9,
10). Adding tcu-PA to normal platelet
releasate resulted in the degradation of
Patients with Quebec platelet disorder have an unusual
biochemical defect that causes their Like normal platelets, QPD platelets store plasma-derived and
megakaryocyte-synthesized proteins within their
u-PA has a number of different forms, and its tcu-PA form has much greater plasminogen-activating activity than uncleaved scu-PA.28-31 These forms can be distinguished from each other and from LMW u-PA using nonreduced and reduced SDS-PAGE.28-31 Whereas normal platelets have been reported to contain mostly scu-PA,23,24 we found QPD platelets contained predominantly active tcu-PA, minimal scu-PA, some LMW u-PA, and a small amount of u-PA in high-molecular weight complexes. Moreover, unlike normal platelets, QPD platelets contained plasminogen that was proteolyzed and that comigrated with plasmin. The high-molecular weight u-PA complexes in QPD platelets resembled the complexes generated by incubating exogenous u-PA with normal releasate, and they included forms recognized by PAI-1 antibodies. These data suggest the very large forms of u-PA in QPD platelets, like the large forms in normal platelets,23,24 represent u-PA complexed to soluble platelet protease inhibitors, such as PAI-132-36 and protease nexin 1.37 The unregulated u-PA activity in QPD platelets indicates they do
not contain sufficient protease inhibitors to fully neutralize their
stored u-PA. Normal platelets contain large amounts of the u-PA
inhibitor PAI-1 within their The diversity of proteins degraded in QPD platelets has suggested
that fairly broad-specificity protease(s) are involved. The FDPs
secreted by QPD platelets are not recognized by a monoclonal antibody
specific for plasmin-degraded fibrinogen.6 Using sensitive Western blots, we observed that QPD platelets contained proteolyzed forms of plasminogen with the mobility of plasmin, but we were unable
to detect plasmin activity in QPD platelet releasates by zymography,
even after u-PA was immunodepleted. tcu-PA and LMW u-PA are known to
proteolyze fibrinogen in addition to plasminogen,42 but
where they cleave fibrinogen has not been determined. Moreover, it is
not yet known whether tcu-PA and LMW u-PA can cleave other potential substrates within platelets. We observed that the net effects
of adding exogenous tcu-PA to normal platelet secretory proteins (in
concentrations similar to the increased u-PA in QPD platelets) were a
loss of intact plasminogen and the proteolysis of many u-PA is normally expressed in many different tissues,45
and it is thought to play a role in diverse physiological and
pathological processes.31 In mice, u-PA deficiency causes
problems with excess fibrin deposition, whereas its overexpression in
the liver results in bleeding, marked hypofibrinogenemia, and systemic
fibrinogenolysis.46,47 The QPD has biochemical
abnormalities distinct from other platelet storage pool
disorders1 and from congenital bleeding disorders associated with increased t-PA levels or t-PA-related proteins in
plasma.48,49 Like The QPD is the only inherited bleeding disorder in humans associated with increased levels of u-PA in blood. Unraveling its genetic cause is likely to provide further insights into this unusual and sometimes fatal bleeding disorder.
C.P.M.H. is the recipient of a Career Investigator Award from the Heart and Stroke Foundation of Ontario, and a Canada Research Chair in Molecular Hemostasis from the Government of Canada and a Premier's Research Excellence Award from the Ontario Government. We thank Dr Jack Henkin at Abbott Laboratories (North Chicago, IL) for the gift of recombinant u-PA.
Submitted November 6, 2000; accepted March 12, 2001.
Supported by grant NA 4379 from the Heart and Stroke Foundation of Ontario (C.P.M.H.) and a grant from Aventis Behring Canada (G.E.R.). W.H.A.K. is the recipient of a Medical Research Council of Canada/Heart and Stroke Foundation of Canada Post-Doctoral Fellowship Award.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Catherine P. M. Hayward, Department of Pathology and Molecular Medicine, McMaster University Medical Center, Rm 2N32, 1200 Main St West, Hamilton, Ontario, Canada L8N 3Z5; e-mail: haywrdc{at}mcmaster.ca.
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© 2001 by The American Society of Hematology.
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  M. Diamandis, A. D. Paterson, J. M. Rommens, D. K. Veljkovic, J. Blavignac, D. E. Bulman, J. S. Waye, F. Derome, G. E. Rivard, and C. P. M. Hayward Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes Blood, February 12, 2009; 113(7): 1543 - 1546. [Abstract] [Full Text] [PDF]
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D. K. Veljkovic, G. E. Rivard, M. Diamandis, J. Blavignac, E. M. Cramer-Borde, and C. P. M. Hayward Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation Blood, February 12, 2009; 113(7): 1535 - 1542. [Abstract] [Full Text] [PDF]
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B. Lo, L. Li, P. Gissen, H. Christensen, P. J. McKiernan, C. Ye, M. Abdelhaleem, J. A. Hayes, M. D. Williams, D. Chitayat, et al. Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet {alpha}-granule biogenesis Blood, December 15, 2005; 106(13): 4159 - 4166. [Abstract] [Full Text] [PDF]
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H. McKay, F. Derome, M. A. Haq, S. Whittaker, E. Arnold, F. Adam, N. M. Heddle, G. E. Rivard, and C. P. M. Hayward Bleeding risks associated with inheritance of the Quebec platelet disorder Blood, July 1, 2004; 104(1): 159 - 165. [Abstract] [Full Text] [PDF]
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H. V. Yarovoi, D. Kufrin, D. E. Eslin, M. A. Thornton, S. L. Haberichter, Q. Shi, H. Zhu, R. Camire, S. S. Fakharzadeh, M. A. Kowalska, et al. Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment Blood, December 1, 2003; 102(12): 4006 - 4013. [Abstract] [Full Text] [PDF]
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T. L. Yang, S. W. Pipe, A. Yang, and D. Ginsburg Biosynthetic origin and functional significance of murine platelet factor V Blood, October 15, 2003; 102(8): 2851 - 2855. [Abstract] [Full Text] [PDF]
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H. Sun, T. L. Yang, A. Yang, X. Wang, and D. Ginsburg The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis Blood, October 15, 2003; 102(8): 2856 - 2861. [Abstract] [Full Text] [PDF]
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D. Kufrin, D. E. Eslin, K. Bdeir, J.-C. Murciano, A. Kuo, M. A. Kowalska, J. L. Degen, B. S. Sachais, D. B. Cines, and M. Poncz Antithrombotic thrombocytes: ectopic expression of urokinase-type plasminogen activator in platelets Blood, August 1, 2003; 102(3): 926 - 933. [Abstract] [Full Text] [PDF]
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 T. J. Podor, D. Singh, P. Chindemi, D. M. Foulon, R. McKelvie, J. I. Weitz, R. Austin, G. Boudreau, and R. Davies Vimentin Exposed on Activated Platelets and Platelet Microparticles Localizes Vitronectin and Plasminogen Activator Inhibitor Complexes on Their Surface J. Biol. Chem., February 22, 2002; 277(9): 7529 - 7539. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-granule protein degradation. The degradation of 
and some of the other