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Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator by Walter H. A.

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Presentation on theme: "Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator by Walter H. A."— Presentation transcript:

1 Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator by Walter H. A. Kahr, Shilun Zheng, Prameet M. Sheth, Menaka Pai, Alison Cowie, Madeleine Bouchard, Thomas J. Podor, Georges E. Rivard, and Catherine P. M. Hayward Blood Volume 98(2): July 15, 2001 ©2001 by American Society of Hematology

2 Fibrinolytic proteases in QPD platelets
Fibrinolytic proteases in QPD platelets.Platelet lysates (lys; 3 μL) and releasates (rel; 3 μL) from patients with QPD (Q) and control subjects (C) were spotted onto fibrin substrate gels. Fibrinolytic proteases in QPD platelets.Platelet lysates (lys; 3 μL) and releasates (rel; 3 μL) from patients with QPD (Q) and control subjects (C) were spotted onto fibrin substrate gels. (A) There was abnormal fibrinolytic activity in QPD platelet lysates and releasates that was blocked by the serine protease inhibitor AEBSF. The secretagogue ionophore A23187 (iono) released more of this activity from QPD platelets than ADP. (B) The fibrinolytic enzymes released by QPD platelets were not inhibited in 1:2 mixtures with normal platelet lysates or releasates (tested ratios of patient ionophore releasate–control samples are shown). Protein rings, without evidence of fibrinolysis, were seen in the tests of control lysates. Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

3 Fibrin gel zymograms of proteases in QPD platelets
Fibrin gel zymograms of proteases in QPD platelets.Samples of QPD (Q1 and Q2 indicate patients from families 1 and 2, respectively) and control (C) platelet lysates (lys; A, 12.5 μL; B, 2 μL) and ADP releasates (ADP rel; A, 50 μL; B, 2 μL) were analyzed on ... Fibrin gel zymograms of proteases in QPD platelets.Samples of QPD (Q1 and Q2 indicate patients from families 1 and 2, respectively) and control (C) platelet lysates (lys; A, 12.5 μL; B, 2 μL) and ADP releasates (ADP rel; A, 50 μL; B, 2 μL) were analyzed on fibrin substrate gels after 9% (A) or 5% to 15% (B) nonreduced SDS-PAGE. (A) Analyses using gels without added plasminogen indicated QPD platelets contained 50-kd (major band) and 100-kd (minor component) secretable, fibrinolytic proteases (arrows) that were not evident in the same amount of control samples. (B) Comparative analyses, using substrate gels with (+) or without (−) added plasminogen, indicated the 100- and 50-kd QPD platelet proteases had properties of plasminogen activators. A 33-kd plasminogen activator was also detected in QPD (but not in control) platelets and releasates (Mr based on 5%-15% nonreduced SDS-PAGE). Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

4 Western blots of u-PA in platelets
Western blots of u-PA in platelets.u-PA in platelet lysates (L; 5 μL) and ionophore releasates (R; 5 μL) from patients with QPD (Q) and healthy control subjects (C) was visualized with monoclonal or polyclonal antisera after 10% SDS-PAGE. Western blots of u-PA in platelets.u-PA in platelet lysates (L; 5 μL) and ionophore releasates (R; 5 μL) from patients with QPD (Q) and healthy control subjects (C) was visualized with monoclonal or polyclonal antisera after 10% SDS-PAGE. (A) Nonreduced analyses indicated QPD platelet lysates and releasates contained abnormally large amounts of u-PA. The predominant form of u-PA in QPD platelets comigrated, nonreduced, with recombinant scu-PA (scuPA, 10 ng), though larger and smaller forms were also detected. When control platelet releasate was incubated (lanes *) with recombinant scu-PA (lane CR + scuPA*), high–molecular weight u-PA complexes were generated that resembled large forms of u-PA in QPD releasates (arrow indicates the most abundant large form). (B) QPD platelet releasates and lysates (R and L, 5 μL nonreduced, 20 μL reduced) contained forms of u-PA with the characteristic nonreduced/reduced mobility of purified tcu-PA (tcuPA; 4.4 ng nonreduced, 17.6 ng reduced) and LMW u-PA (LMWuPA; 3.2 ng nonreduced, 12.8 ng reduced). Arrows indicate the A chain (A*) of reduced tcu-PA, the B chain (B*) common to reduced tcu-PA and LMW u-PA, and the nonreduced form of LMW u-PA (LMW). A reduced protein with the mobility of the A chain of tcu-PA was seen in prolonged exposures of the QR and QL lanes (not shown). Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

5 Plasminogen activators in QPD platelets
Plasminogen activators in QPD platelets.Plasminogen activators were analyzed on casein substrate gels containing plasminogen. Plasminogen activators in QPD platelets.Plasminogen activators were analyzed on casein substrate gels containing plasminogen. (A, D) Samples tested after 10% nonreduced SDS-PAGE. (B, C) Samples spotted directly onto substrate gels. (A) QPD releasate (QR, 6 μL; QR*, 10 μL) contained plasminogen activators that comigrated with tcu-PA (tcuPA, 8.8 ng) and LMW u-PA (LMWuPA, 3.2 ng), but not with purified plasmin (10 ng) or t-PA (tPA, 1 IU). (B) Unlike t-PA (0.5 IU), tcu-PA (5 ng) and the plasminogen activators in 1 μL QR and QL (QPD lysate) were inhibited on gels with added (+) 1 mM amiloride. (C) Large amounts of recombinant PAI-1 (final concentrations shown) were required to fully neutralize the plasminogen activators in pooled QR. (D) Zymograms indicated that the 100-, 50-, and 33-kd proteases in QR (5 μL/lane) were removed by rabbit antibodies to human u-PA (depl), but not by normal rabbit IgG (sham). The 33-kd protease in lanes sham and QR was evident on the original gel. Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

6 Platelet u-PA and β-actin mRNA, analyzed by RT-PCR
Platelet u-PA and β-actin mRNA, analyzed by RT-PCR.u-PA and β-actin transcripts were amplified separately before running the products in lanes of the gel. Platelet u-PA and β-actin mRNA, analyzed by RT-PCR.u-PA and β-actin transcripts were amplified separately before running the products in lanes of the gel. Lanes compare transcripts amplified from platelets of 2 controls (C) and 3 patients with the QPD (Q), with transcripts from TPA-stimulated K562 cells. QPD platelets contained normal amounts of β-actin mRNA and increased u-PA mRNA, which was not detectable in control platelets. Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

7 PAI-1 in control and QPD platelets
PAI-1 in control and QPD platelets.(A) PAI-1 in control (C) and QPD (Q; 3 patients [Pt] are shown) samples was analyzed by Western blotting with rabbit anti–human PAI-1 after 10% nonreduced SDS-PAGE. PAI-1 in control and QPD platelets.(A) PAI-1 in control (C) and QPD (Q; 3 patients [Pt] are shown) samples was analyzed by Western blotting with rabbit anti–human PAI-1 after 10% nonreduced SDS-PAGE. Lanes compare 5 μL lysate (L) and ionophore releasates (R), 20 μL K562 media, and 5 μL control ionophore releasate, incubated with 0 to 400 ng/mL tcu-PA (CR + tcuPA), as indicated. Lane * shows the PAI-1 affinity purified from QL using monoclonal anti–u-PA. QPD platelet lysates contained PAI-1 in high–molecular weight complexes (bands near the 98-kd marker) that comigrated with the complexes generated by adding tcu-PA to control releasates. Proteolyzed PAI-1 (arrow) was detected in QPD platelet lysates and in long exposures (not shown) of control releasates incubated with tcu-PA. (B) u-PA–PAI-1 complex ELISA indicated that, unlike control samples, QPD platelet lysates and releasates were unable to generate additional u-PA–PAI-1 complexes in vitro with added (+) tcu-PA (data representative of 2 separate experiments with pooled samples). Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

8 Plasminogen in control and QPD platelets
Plasminogen in control and QPD platelets.Proteins were analyzed by Western blotting with rabbit anti–human plasminogen after 10% reduced SDS-PAGE. Plasminogen in control and QPD platelets.Proteins were analyzed by Western blotting with rabbit anti–human plasminogen after 10% reduced SDS-PAGE. Lanes compare 12 μL control (C) and QPD (Q) lysates (L) and ionophore releasates (R) and control ionophore releasate, incubated overnight with 0 to 400 ng/mL tcu-PA (CR + tcuPA), as indicated. As references, Glu-plasminogen (plg; 20 ng/lane) and the plasmin generated by digesting Glu-plasminogen (60 ng) with 100 ng/mL tcu-PA (plg + tcuPA) are shown. QPD platelets contained proteolyzed plasminogen that comigrated with the heavy chain of plasmin (arrow; the light chain of plasmin was not visualized by the antisera). They also contained correspondingly reduced proportions of intact plasminogen and some smaller proteolyzed components, not evident in normal platelets. When control platelet releasate was incubated with exogenous tcu-PA, there was a similar loss of intact plasminogen, but the extent of plasminogen proteolysis was not as complete as in QPD platelets. Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

9 α-granule fibrinogen and fibronectin degradation
α-granule fibrinogen and fibronectin degradation.Western blots compare nonreduced fibrinogen (A; 5%-8% SDS-PAGE) and fibronectin (B; 4%-8% SDS-PAGE) in QPD (Q) and control (C) platelet lysates with the degradation products generated by incubating normal pla... α-granule fibrinogen and fibronectin degradation.Western blots compare nonreduced fibrinogen (A; 5%-8% SDS-PAGE) and fibronectin (B; 4%-8% SDS-PAGE) in QPD (Q) and control (C) platelet lysates with the degradation products generated by incubating normal platelet ionophore releasate (CR) with 0, 25, 50, or 100 ng/mL tcu-PA (tcuPA), as indicated (volumes of releasate and lysates: A, 3.6 μL/lane; B, 15 μL/lane). When tcu-PA was incubated with normal platelet secretory proteins, α-granule fibrinogen and fibronectin were proteolyzed to forms that comigrated (arrows) with their degraded forms in QPD platelets. Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

10 α-granule thrombospondin-1, von Willebrand factor, and osteonectin degradation.Western blots compare thrombospondin-1 (A), von Willebrand factor (vWF) (B), and osteonectin (C) in QPD platelet lysates with degraded forms generated by incubating normal platel... α-granule thrombospondin-1, von Willebrand factor, and osteonectin degradation.Western blots compare thrombospondin-1 (A), von Willebrand factor (vWF) (B), and osteonectin (C) in QPD platelet lysates with degraded forms generated by incubating normal platelet ionophore releasate with tcu-PA (lanes and samples as in Figure 8; ng/mL tcu-PA are indicated). (A) When tcu-PA was incubated with normal platelet releasate, α-granule thrombospondin-1 was partially proteolyzed to a form that comigrated with the larger thrombospondin-1 degradation product in QPD platelets (arrow) (lanes compare 24 μL releasate and lysate, after reduced 4%-8% SDS-PAGE). (B) tcu-PA induced the degradation of von Willebrand factor to a form that comigrated with degraded von Willebrand factor in QPD platelets (arrow) (lanes compare 6 μL of releasate and lysate, after reduced 7% SDS-PAGE). (C) tcu-PA also induced the proteolysis of α-granule osteonectin, generating a form that comigrated with degraded osteonectin in QPD platelets (arrow) (lanes compare 9 μL releasate and lysate, after reduced 12% SDS-PAGE). Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology

11 α-granule factor V and multimerin degradation
α-granule factor V and multimerin degradation.Western blots compare proteins in QPD (Q) and control (C) platelet lysates (samples with all inhibitors) with the forms generated by incubating normal platelet ionophore releasate (CR; A) or lysate (CL; B, lysat... α-granule factor V and multimerin degradation.Western blots compare proteins in QPD (Q) and control (C) platelet lysates (samples with all inhibitors) with the forms generated by incubating normal platelet ionophore releasate (CR; A) or lysate (CL; B, lysate without serine protease inhibitors) with 0, 25, 50, or 100 ng/mL tcu-PA (tcuPA), as indicated. (A) When u-PA was incubated with normal platelet secretory proteins, α-granule factor V was degraded, resulting in a loss of factor V detectable by Western blotting with polyclonal antisera, as in QPD platelets (lanes compare 18 μL lysate and releasate, after reduced 4%-8% SDS-PAGE). (B) Although there was some loss of detectable multimerin in lysates incubated without tcu-PA, tcu-PA reduced the detectable multimerin, as in QPD platelets (lanes compare 15 μL lysates, separated on nonreduced multimer gels and probed with a mixture of monoclonal and polyclonal antimultimerin). Traces of degraded multimerin (bands below the smallest multimerin polymer in normal platelets) were evident in the u-PA digests (B) and in QPD platelets, with longer exposures (not shown). Walter H. A. Kahr et al. Blood 2001;98: ©2001 by American Society of Hematology


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