SUMMARY1. A new technique was developed for the measurement of extracellular free ATP in very small samples of whole blood using the luciferin-luciferase enzyme system. The method had a very low background corresponding to approximately 10-16 mol ATP.2. ATP was measured in blood as it emerged during haemostasis following precise puncture of rat and rabbit arteries and after standardized incisions of human skin by the Simplate device.3. The initial concentration of free ATP in blood emerging 2-4 s after vascular injury was about 2 x 1O-7 M in rats and rabbits and about 2 x 10-6 M in humans.4. The free-ATP concentration increased to 2 x 10-5 M 3-5 min after injury and these increases could be prevented by heparin (20 u./ml).5. The source of the initial free ATP was identified as damaged cells in the injured vessel wall. Sufficient ADP, both released as such with ATP and generated by enzymic dephosphorylation of ATP, would be present at the site of injury to initiate haemostatic aggregation of platelets.

Primary haemostasis was simulated in vitro under standardized geometrical, rheological and biochemical conditions. The system was based on the hypothesis that adenine nucleotides, especially adenosine 5′-diphosphate, released from injured vessel wall cells play an important role for the initial platelet aggregation following lesion of small vessels. The device allows the reproducible measurement of in vitro bleeding time and volume in small samples of whole blood.

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to α-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with α-actinin-1 along endothelial actin stress fibers. In contrast to α-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion proteins to stress fibers in endothelial cells. Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 of α-actinin-1. The study further shows that CLP36 binds to α-actinin in resting platelets and translocates as a CLP36/α-actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via α-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells. The study suggests that CLP36 may direct α-actinin-1 to specific actin structures and at this position might modulate the function of α-actinin-1.

A new technique, which simulates primary haemostasis in vitro was tested using blood from control persons and from patients with a defined abnormality of primary haemostasis. The device allows the reproducible measurement of in vitro bleeding time and volume in small samples of whole blood. Inhibition of platelet adhesion and aggregation can very sensitively be detected with the new technique, which allows its possible use in clinical or pharmacological applications.

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to α-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with α-actinin-1 along endothelial actin stress fibers. In contrast to α-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion proteins to stress fibers in endothelial cells. Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 of α-actinin-1. The study further shows that CLP36 binds to α-actinin in resting platelets and translocates as a CLP36/α-actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via α-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells. The study suggests that CLP36 may direct α-actinin-1 to specific actin structures and at this position might modulate the function of α-actinin-1.