Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.To promote effective hemostasis, platelets must respond quickly to changes in normal blood flow or vessel injury (1, 2). After vascular injury, platelets adhere to exposed subendothelium, aggregate, and form a primary platelet plug. Platelet activation and initiation of coagulation follow with stabilization of the platelet plug by the formation of fibrin. The initiation of a thrombus at a site of vascular injury is mediated through platelet membrane glycoprotein (GP) receptors (3, 4). Platelet adhesion to a damaged vessel wall and its extracellular matrix at high shear is primarily mediated through the specific interaction of the platelet membrane GPIb-IX complex and bound von Willebrand factor (vWF) (5-7), which is synthesized and released into plasma and the vessel wall by endothelial cells (1). Platelet adhesion at low shear rates is mediated by several interactions, including collagen with the a2131 integrin (7). Platelet adhesion stimulates a spreading of the platelet (8).Although the mechanism of platelet spreading has not been completely characterized, recent in vitro studies have shown that platelets will spread on surfaces coated with fibrinogen (9) or polymerized fibrin (10). The activation of the GPIIb/IIIa receptor by agents such as ADP results in a conformational change in the receptor (11-13). The activated receptor binds fibrinogen, which forms a "bridge" between the platelets, and causes aggregation (1,14,15). Activated platelets provide the phospholipid surface for the assembly of blood clotting enzyme complexes, and the concentration and localization of activated coagulant proteins at sites of vessel wall injury may be facilitated by adherent platelets (16). Internal storage granules in platelets release clot-promoting contents in response to activation of bi...

We examined the myofibril biochemical, structural, and biophysical properties of C2C12, a mouse skeletal muscle cell line (American Type Culture Collection), to assess whether force development and the sensitivity of the myofilaments to calcium could be measured in C2C12 myotubes and whether a cardiac contractile protein, troponin T, is expressed and incorporated into C2C12 myofibrils. When myoblasts fused and differentiated into myotubes, expression of myofilament proteins was initiated. Multiple cardiac and skeletal muscle troponin T isoforms were coexpressed. Cardiac troponin T expression increased and then decreased with time. Fluorescence immunocytochemistry demonstrated incorporation of cardiac troponin T isoforms into the myofibrils. At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns). The estimated maximum force developed by chemically skinned myotubes at 6-7 days poststarvation, 0.88 +/- 0.12 microN (mean +/- 95% confidence interval, n = 5), was significantly less (P < 0.05) than that at 10-13 days poststarvation, 1.12 +/- 0.12 microN (n = 7). The force-pCa relation yielded a Hill coefficient of 2.9 +/- 0.6 (n = 7) and half-maximal activation at pCa of 5.77 +/- 0.20. The demonstration that the biophysical properties of C2C12 cells can be measured and that cardiac and skeletal muscle troponin T isoforms are incorporated and colocalized into myofibrils suggest that these cells could be a useful model to assess the effects of exogenous native and mutated cardiac and skeletal contractile protein isoforms on myofilament function.

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In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive

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