Background

Rest Hemostasis Thrombosis

CD39
NO

Flow Flow

ADP TxA2
PGI2

Thrombin
Tissue Factor Tissue Factor Tissue Factor

Platelet Activation Activating Endothelium

Platelets
Inhibitory Vessel wall 1

Platelets stop bleeding, but unwarranted activation causes thrombosis

 Background
Thrombopoiesis Homeostasis Clearance

Hepatocytes

Macrophages

Circulating
Megakaryocyte Platelets
Hemostasis/
thrombosis

Average platelet lifespan is ~7-10 days in humans and ~4-5 days in mice
 Background
Thrombopoiesis Homeostasis Clearance

Splenectomy
Cancer
ET Hepatocytes

Macrophages

Circulating
Megakaryocyte Platelets
Hemostasis/
thrombosis

Pathological states resulting in increased production lead to thrombocythemia

 Background
Thrombopoiesis Homeostasis Clearance

Leukemia Immune
Trauma Hepatocytes
BMF

Macrophages

Circulating
Megakaryocyte Platelets
Hemostasis/
thrombosis

Decreased production or greater clearance leads to thrombocytopenia

 Background
Room Temp (~22° C) Refrigerated (4° C)

~5 d >10 d

In vitro In vivo In vitro In vivo

Murphy, S. and F. H. Gardner. 1969. NEJM. 280: 1094-1098.

Goal: what mechanism(s) lead to cold-induced clearance of platelets (PLTs)

How do chilled PLTs function and where do they go?

EGTA: blocks Ca2+ (important for activation)

Cytochalasin B: blocks actin (contractile filaments)

Chilled PLT clearance is NOT due to shape change

 Light Transmission Aggregometry

Grove, Erik Lerkevang. Danish medical journal. 59 9 (2012): B4506.

Light transmission aggregometry is the “gold standard” platelet function test

How do chilled PLTs function and where do they go?

EGTA: blocks Ca2+ (important for activation)

Cytochalasin B: blocks actin (contractile filaments)

Chilled PLT clearance is NOT due to shape change or impaired function

How do chilled PLTs function and where do they go?

Chilled PLT clearance occurs in the liver and is mediated by macrophages

 What is required for cold-induced clearance?
vWf: platelet-interacting protein C3: promotes phagocytosis CR3: liver macrophage receptor

WT platelets  vWF-/- mouse WT platelets  C3-/- mouse WT platelets  CR3-/- mouse

Chilled PLT clearance is mediated by macrophage CR3 (aMb3) receptors

 What is required for cold-induced clearance?
Isolate PLTs

Label @
4° or 22°
and mix

Expose liver

Infuse PLTs
Incubate @
intervals

Image PLTs

Supports hypothesis that chilled PLTs are cleared by liver CR3 receptors
 GPIb-IX-V complex

GPIb-IX-V is the main receptor for vWF and aids PLT adhesion
Is GPIb involved in clearance of chilled platelets?

Clearance of chilled PLTs is mediated by GPIb complex and CR3

 Is GPIb involved in clearance of chilled platelets?

Chilling PLTs does NOT increase vWF binding but causes GPIb clustering
 Is GPIb involved in phagocytosis of chilled PLTs?
Isolate PLTs
Label @ • THP-1: phagocytic human
4° or 22° monocyte cell line
Incubate w/
THP-1 cells • Mocarhagin (Moc): cobra
venom metalloproteinase
30 min that cleaves GPIba
Remove
adherent
PLTS
Differentiated cells =
>CR3 expression
Remove THP-1
and do FACS

GPIb complex mediates phagocytosis of chilled human PLTs in vitro

Does chilling affect other aspects of PLT physiology?
• Hemostasis
• Does NOT change the expression of hemostatic receptors
• Does NOT increase markers of platelet activation

• Apoptosis
• Does NOT increase phosphatidylserine exposure

• Immunity
• Does NOT increase the binding of immunoglobulins

Chilling PLTs does NOT affect hemostasis, apoptosis, or immune responses

Does chilling in vivo induce PLT clearance?

Clearance of PLTs occurs in chilled mice and is CR3-dependent

 Model for Basal Clearance of PLTs

PLTs not required for hemostasis are cleared by phagocytosis

 Summary
• Clearance of chilled PLTs is NOT due to shape change or activation

• Chilled PLTs are cleared in the liver by macrophages

• Clearance depends on macrophage CR3 (aMb3) receptors

• Clearance depends on platelet GPIb-IX-V receptors

• Temperature changes may prime platelets for clearance in vivo

 Questions
• How might this information be applied in a clinical setting?
• Pre-treat recipients with CR3-inhibitor
• Treat donor platelets with GPIb inhibitor

• What are potential complications?

• CR3 may be involved in phagocytosis of other cells
• Blocking GPIb may interfere with hemostasis
 Current State of the Art
• RT-PLTs
• Maintain normal hemostatic function with minimal off-target effects
• BUT can still only be stored ~5 days, meaning lots of waste

• COLD-PLTs
• Are more activated but can provide enhanced hemostatic function
• Can be stored longer, reducing waste, but only good for acute use

• CRYO-PLTs
• Form platelet microparticles, with enhanced hemostatic function
• Can be stored much longer, but also only good for acute use

RT-PLTs are still standard but COLD- and CRYO-PLTs useful for trauma
 Current Models of PLT Clearance

AMR clears desialylated PLTs in vivo; chilled PLTs cleared by AMR and aMb2
Figure 6A-D
Figure 6E
 Table 1

Activation
Apoptosis
Immunity