Blood, Vol. 112, Issue 7, 2810-2816, October 1, 2008

Polyphosphate enhances fibrin clot structure
Blood Smith and Morrissey 112: 2810

Supplemental materials for: Smith and Morrissey

Files in this Data Supplement:

• Figure S1. Ca²⁺ promotes the ability of polyP to enhance clot turbidity, while Zn²⁺, Mg²⁺ or Mn²⁺ do not (JPG, 35.5 KB) -
  Reactions contained 2.6 mg/mL fibrinogen in TBS plus the indicated metal ion concentrations. The fibrinogen/metal ion mixtures were preincubated for 15 minutes with (▼) or without (○) 1 mM polyP, after which clotting was initiated with 1 nM thrombin. (A-C) Turbidity of clots formed in the absence of CaCl₂. (D-F) Turbidity of clots formed in the presence of 2.5 mM CaCl₂. In panels A-C (no calcium), none of the tested metal ions supported the ability of polyP to enhance fibrin clot turbidity, although Zn²⁺ and Mn²⁺ enhanced clot turbidity in a polyP-independent fashion. In panels E and F, supraphysiologic concentrations of Mg²⁺ or Mn²⁺ antagonized the ability of polyP to enhance fibrin turbidity in the presence of 2.5 mM Ca²⁺.
  
  
  
  
  
  
• Figure S2. The effect of polyP on clot turbidity is enhanced by pre-incubation of fibrinogen, Ca²⁺, and polyp (JPG, 16.3 KB) -
  Final reaction mixtures contained 2.6 mg/mL fibrinogen in TBS plus 2.5 mM CaCl₂, either with 1 mM polyP (black bars) or without polyP (white bars), although the order of addition of Ca²⁺ and polyP was varied. This was accomplished by incubating the indicated components with either the fibrinogen or the thrombin for 15 minutes, after which clotting was initiated by mixing together the fibrinogen- and thrombin-containing solutions. (In all cases, the final thrombin concentration was 1 nM.) For the black bars: In reaction A, both CaCl₂ and polyP were preincubated with fibrinogen. In reaction B, CaCl₂ was preincubated with fibrinogen, while polyP was preincubated with thrombin. In reaction C, polyP was preincubated with fibrinogen, while CaCl₂ was preincubated with thrombin. In reaction D, both CaCl₂ and polyP were incubated with thrombin. Data are mean ± standard error (n=4). Preincubation of all three (fibrinogen, Ca²⁺ and polyP) were required for maximal enhancement of clot turbidity.
  
  
  
  
  
  
• Figure S3. The effect of polyP on turbidity is not dependent on factor XIII(a) cross-linking activity (JPG, 13.1 KB) -
  Clotting reactions contained 2.6 mg/mL fibrinogen in TBS plus 2.5 mM CaCl₂ and the indicated additives, either with 1 mM polyP (black bars) or without polyP (white bars). Clotting was initiated with 1 nM thrombin. Reaction A contained the small amount of factor XIII that was already present in the fibrinogen preparation. Reaction B contained 1 mM iodoacetamide (a factor XIIIa inhibitor), while reaction C contained 100 nM added factor XIII. (In reactions B and C, the iodoacetamide or factor XIII were added to the fibrinogen ± polyP at the start of the 15 minute preincubation period prior to thrombin addition). Reaction D contained 100 nM factor XIII added to the thrombin component so it would be preactivated to factor XIIIa before being mixed with fibrinogen. Data are mean ± standard error (n=4). PolyP enhanced fibrin clot turbidity in all cases, indicating that its action is not dependent on factor XIII(a) activity.