Method for determining the functional activity of free protein S or protein C in a plasma sample

A method for determining in a human plasma sample containing 0.02 to 50 percent plasma by volume the functional activity of free Protein S or Protein C, said method comprising:

• (i) adding to said plasma sample a snake venom enzyme which activates protein C or activated Protein C when Protein S is to be determined, or a snake venom enzyme which activates protein C and Protein S when Protein C is to be determined,
• (ii) adding (a) coagulation enzyme FIX.sub.a in an amount of 1.times.10.sup.-15 to 1.times.10.sup.-6 mol/l, (b) at least one further coagulation reagent selected from the group consisting of cofactor FV.sub.a in an amount of 1.times.10.sup.-12 to 1.times.10.sup.-9 mol/l and phospholipids in an amount of 1.times.10.sup.-6 to 3.times.10.sup.-4 mol/l, (c) calcium ions in an amount of 10.sup.-4 to 5.times.10.sup.-2 mol/l and (d) a buffer to maintain a pH of 6.5 to 9.5 and having an ion strength of 0 to 0.6, to form a mixture and incubating said mixture to form thrombin from prothrombin,
• (iii) measuring the quantity of thrombin formed;
• and
• (iv) determining the content of functionally active Protein S or functionally active Protein C in the original plasma sample on the basis of the measured quantity of thrombin by comparing the measured quantity with a standard;
• the content of Protein S or Protein C being inversely related to the quantity of thrombin formed.