Changes in ligand binding ability of the integrin alpha IIb beta 3 can be monitored by the concomitant expression of ligand-inducible binding sites (LIBS). A new LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of beta 3 recognized by the murine monoclonal antibody (mAb) AP5, is sensitive both to the binding of ligand and to micromolar differences in divalent cation levels. Calcium or magnesium can completely inhibit the binding of AP5 to alpha IIb beta 3 on platelets, with ID50 values of 80 and 1500 microM, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the presence of 1 mM calcium plus 1 mM magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The unique location of the AP5 LIBS was determined by comparing the binding of LIBS-specific mAb to recombinant human-Xenopus beta 3 chimeras produced in a baculovirus expression system. AP5 defines one region at the amino terminus beta 3 1-6. A second region, defined by mAb D3GP3, is probably located within beta 3 422-490, confirming the finding of Kouns et al. (Kouns, W. C., Newman, P.J., Puckett, K. J., Miller, A. A., Wall, C. D., Fox, C. F., Seyer, J. M., and Jennings, L. K. (1991) Blood 78, 3215-3223). The third region, encompassing at most residues 490-690, and perhaps more precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2, anti-LIBS3, anti-LIBS6, and P41. Since its exposure is uniquely regulated by both divalent cations and ligand, the amino terminus of beta 3 may be involved in control of ligand binding by divalent cation mobilization.

The actin dependence of the rate and magnitude of the initial phosphate burst was measured using both quench-How and stopped-flow kinetic techniques. These studies revealed that even at high actin concentrations the magnitude of the phosphate burst was a significant fraction of the magnitude that exists in the absence of actin. Furthermore, it was shown that the rate of the burst rises rapidly as a function of the actin concentration. Detailed modeling with the four-state model revealed that if the predicted V^ is constrained to be approximately equal to the extrapolated value (double reciprocal plot) and if the apparent dissociation constant of subfragment-1 to actin divided by the apparent activation constant of the actin-activated myosin ATPase activity (K^^^/K ArPmK ) is constrained to be considerably different from one, then the model is unable to simultaneously account for the ATPase activity and the rate and magnitude of the initial inorganic phosphate burst. (Circulation Research 1989;65:515-525) R ecent studies on the steady-state properties of porcine cardiac subfragment-1 (S-l) 1 have revealed that the biochemical kinetics of cardiac S-l are very similar to the kinetics of rabbit skeletal S-l.2 It was shown that ^bindmgj the apparent dissociation constant of S-l to actin, and /CATP»K> the apparent activation constant of the actin-activated myosin ATPase activity, of porcine cardiac S-l were, within experimental error, equal to those of skeletal S-l and that the ratio of these constants (i.e., Ktinding/iCATp^) for the cardiac proteins is approximately 5 : 1. The only significant difference between skeletal and cardiac S-l was that the extrapolated V^, of the double reciprocal plot 3 was approximately 2/sec for cardiac S-l and 4-5/sec for skeletal S-l at 15° C and low ionic strength (0.013 M). Furthermore, it was shown that cross-linked actoS-1, prepared using the zero-length cross-linking agent l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), possesses a significant ATPase activity. 4 ' 5 This lack of significant inhibition of the ATPase activity of cardiac actoS-1 at saturating actin con- centrations was reported previously for skeletal S-l, and detailed kinetic modeling revealed that a "nondissociating pathway" for ATP hydrolysis was required to account for the data. 5 It was then concluded that the four-state model was the minimal model that could account for the data (see Figure I).1 Because the steady-state kinetics of cardiac S-l are almost identical to rabbit skeletal S-l, except for a significantly different V^, it becomes interesting to pursue the kinetic mechanism of this difference. The important question is whether a four-state model can adequately account for the cardiac data or whether a more complex model (e.g., a six-state model) needs to be postulated. In the current work, we have expanded our prior kinetic studies of cardiac S-l to include presteady-state measurements of the rate and magnitude of the initial phosphate burst, both in the presence and absence of actin. As wa...

We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.

The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.