We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s–1-1000 s–1 in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets “activated” with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s–1. The collision capture efficiency (αG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing ≤10 molecules of 10 million Da) required to yield high capture efficiencies (αG = 0.38 ± .02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with αG = 0.08 ± .01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-IIIa receptor decreased αG by 95 ± 3% (n = 3), while a monoclonal antibody to the vWF site on GPIb caused a 49 ± 7% (n = 8) decrease in αG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIIa receptor to yield stable attachment.
1 We acknowledge the Medical Research Council of Canada for research and personnel support.
References
1
Savage B,
Salvidar E,
Ruggeri ZM.
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
2
Lankhof H,
Wu YP,
Vink T,
Schiphorst ME,
Zerwes HG,
de Groot P,
Sixma JJ.
Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor. Blood 1995; 86: 1035-42.
3
Ikeda Y,
Handa M,
Kamata T,
Kawano K,
Kawai Y,
Watanabe K,
Kawakami K,
Sakai K,
Fukuyama M,
Itagaki I.
et al.
Transmembrane calcium influx associated with von Willebrand factor binding to GPIb in the initiation of shear-induced platelet aggregation. Thromb Haemost 1993; 69: 496-502.
4
Moake JL,
Turner NA,
Stathopoulos NA,
Nolasco LH,
Hellums JD.
Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate and is resistant to aspirin. Blood 1988; 71: 1366-74.
6
Gralnick HR,
Williams SB,
Coller B.
Fibrinogen competes with von Willebrand factor for binding to the Glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin. Blood 1984; 64: 797-800.
8
Goto S,
Salomon DR,
Ikeda Y,
Ruggeri ZM.
Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Willebrand factor to platelets. J Biol Chem 1995; 270: 23352-61.
9
Ruggeri ZM,
De Marco L,
Gatti L,
Bader R,
Montgomery RR.
Platelets have more than one binding site for von Willebrand factor. J Clin Invest 1983; 72: 1-12.
10
Taylor AD,
Neelamegham S,
Hellums JD,
Smith CW,
Simon SI.
Molecular dynamics of the transition from L-selectin to β2-integrin dependent neutrophil adhesion under defined hydrodynamic shear. Biophys J 1996; 71: 3488-500.
11
Frojmovic MM,
Kasirer-Friede A,
Goldsmith HL,
Brown EA.
Surface-secreted von Willebrand factor mediates aggregation of ADP-activated platelets at moderate shear stress: facilitated by GPIb but controlled by GPIIb-IIIa. Thromb Haemost 1997; 77: 568-76.
12
Goldsmith HL,
Frojmovic MM,
Braovac S,
McIntosh F,
Wong T.
Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. III. Shear and extrinsic fibrinogen-dependent effects. Thromb Haemost 1994; 71: 78-90.
13
Goto S,
Ikeda Y,
Salvidar E,
Ruggeri ZM.
Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest 1998; 101: 479-86.
14
Plow EF,
Pierschbacher MD,
Ruoslahti E,
Marguerie GA,
Ginsberg MH.
The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci USA 1985; 82: 8057-61.
16
Schor K,
Darius H,
Matzky R,
Ohlendort R.
The antiplatelet and cardiovascular actions of a new carbaxylic derivative (ZK 36374)-equipotent to PGI2 in vitro. Arch Pharm 1981; 316: 252-5.
17
Coller BS,
Peerschke EI,
Scudder LE,
Sullivan CA.
Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets; additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110.
18
Coller BS,
Peerschke EI,
Scudder LE,
Sullivan CA.
A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest 1983; 72: 325-38.
19
Shattil SJ,
Cunningham M,
Hoaxie JA.
Detection of activation platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-15.
20
Handa M,
Titani K,
Holland LZ,
Roberts JR,
Ruggeri ZM.
The von Willebrand factor-binding domain of platelet membrane glycoprotein GPIb. J Biol Chem 1986; 261: 12579-85.
21
Weinstein M,
Vosburgh E,
Phillips M,
Turner N,
Chute-Rose L,
Moake J.
Isolation from commercial aurin tricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers. Blood 1991; 78: 2291-8.
22
Girma JP,
Fressinaud E,
Christophe O,
Roualt C,
Obert B,
Takahashi Y,
Meyer D.
Aurin tricarboxylic acid inhibits platelet adhesion to collagen by binding to the 509-695 disulphide loop of von Willebrand factor and competing with glycoprotein Ib. Thromb Haemost 1992; 68: 707-13.
25
Zaleski A,
Henriksen RA.
Visualization of the multimeric structure of von Willebrand factor using a peroxidase-conjugated second antibody. J Lab Clin Med 1986; 107: 172-5.
26
Tang SS,
Frojmovic MM.
The effects of pCO2 and pH on platelet shape change and aggregation for human and rabbit platelet-rich plasma. Thromb Res 1977; 10: 135-45.
27
The TH,
Feltkamp TEW.
Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunol 1970; 18: 865-73.
28
Xia Z,
Wong T,
Liu Q,
Kasirer-Friede A,
Brown E,
Frojmovic MM.
Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation. Br J Haematol 1996; 93: 204-14.
29
Williams SB,
McKeown LP,
Krutzch H,
Hansmann Gralnick H.
Purification and characterization of human platelet von Willebrand factor. Br J Haematol 1994; 88: 582-91.
30
Frojmovic MM,
Mooney RF,
Wong T.
Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration. Biophys J 1994; 67: 2060-8.
31
Xia A,
Frojmovic MM.
Aggregation efficiency of activated normal or fixed platelets in a simple shear field: Effect of shear and fibrinogen occupancy. Biophys J 1994; 66: 2190-201.
33
Harrison RL,
McKee PA.
Comparison of thrombin and ristocetin in the interaction between von Willebrand factor and platelets. Blood 1983; 62: 346-53.
34
Azzam K,
Cissé-Thiam M,
Drouet L.
The antithrombotic effect of aurin tricarboxylic acid in the guinea pig is not solely due to the interaction with the von Willebrand factor-GPIb axis. Thromb Haemost 1996; 75: 203-10.
35
Greco NJ,
Jones GD,
Tandon NN,
Kornhauser R,
Jackson B,
Jamieson GA.
Differentiation of the two forms of GPIb functioning as receptors for alpha-thrombin and von Willebrand factor: Ca2+ responses of protease-treated human platelets activated with alpha thrombin and the tethered ligand peptide. Biochem 1996; 35: 915-21.
36
Siedlicki CA,
Lestini BJ,
Kottke-Marchant K,
Eppell SJ,
Wilson DL,
Marchant RE.
Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood 1996; 88: 2939-50.
37
Lawrence MB,
Springer TA.
Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion to integrins. Cell 1991; 65: 859-73.
38
Ikeda Y,
Handa M,
Kawano K,
Kamata T,
Murata M,
Araki Y,
Anbo H,
Kawai Y,
Watanabe K,
Itagaki I,
Sakai K,
Ruggeri Z.
The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest 1991; 87: 1234-40.
39
Ozaki Y,
Satoh K,
Yatomi Y,
Miura S,
Fujimura Y,
Kume S.
Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor. Biochim Biophys Act 1995; 1243: 482-8.
43
Bell DN,
Spain S,
Goldsmith HL.
The ADP-induced aggregation of human platelets in flow through tubes: I. Measurement of the concentration and size of single platelets and aggregates. Biophys J 1989; 56: 817-28.
44
Bell DN,
Spain S,
Goldsmith HL.
The ADP-induced aggregation of human platelets in flow through tubes: II. Effect of shear rate, donor sex, and ADP concentration. Biophys J 1989; 56: 829-43.
45
Frojmovic MM,
Mooney RF,
Wong T.
Dynamics of platelet glycoprotein IIb-IIIa expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation. Biophys J 1994; 67: 2069-75.
47
Heilmann E,
Hourdille P,
Provost A,
Papponneau A,
Nurden AT.
Thrombin-induced platelet aggregates have a dynamic structure. Arterioscl and Thrombos 1991; 11: 704-18.
48
Dong JF,
Hyun W,
Lopez JA.
Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GP Ib alpha. Blood 1995; 86: 4175-83.
49
Berger G,
Massé JM,
Cramer EM.
Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX and V. Blood 1996; 87: 1385-95.
